U.S. patent number 9,139,842 [Application Number 13/208,960] was granted by the patent office on 2015-09-22 for methods and compositions for targeting sequences of interest to the chloroplast.
This patent grant is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. The grantee listed for this patent is Henrik Albert, Linda A. Castle, Matthew Heckert, Jian Lu, Daniel L. Siehl, Yumin Tao. Invention is credited to Henrik Albert, Linda A. Castle, Matthew Heckert, Jian Lu, Daniel L. Siehl, Yumin Tao.
United States Patent |
9,139,842 |
Albert , et al. |
September 22, 2015 |
Methods and compositions for targeting sequences of interest to the
chloroplast
Abstract
Chimeric polynucleotides comprising a nucleotide sequence
encoding a chloroplast transit peptide operably linked to a
heterologous polynucleotide of interest are provided, wherein the
chloroplast transit peptide comprises an amino acid sequence having
the chloroplast transit peptide sequence as set forth in SEQ ID
NO:1 or a biologically active variant or fragment thereof or
wherein the chloroplast transit peptide comprises the sequence set
forth in SEQ ID NO: 58 or an active variant or fragment thereof.
Chimeric polypeptides encoding the same, as well as, cells, plant
cells, plants and seeds are further provided which comprise the
chimeric polynucleotides. Compositions further include HPPD
polypeptides and polynucleotides encoding the same as set forth in
SEQ ID NOS: 57 and 60 or active variants and fragments thereof.
Such sequences comprise the chloroplast transit peptide as set
forth in SEQ ID NO: 58 or an active variants or fragments thereof.
Cells, plant cells, plants and seeds are further provided which
comprise such sequences. Methods of use of the various sequences
are also provided.
Inventors: |
Albert; Henrik (Alameda,
CA), Castle; Linda A. (Mountain View, CA), Heckert;
Matthew (Union City, CA), Lu; Jian (Union City, IA),
Siehl; Daniel L. (Menlo Park, CA), Tao; Yumin
(Urbandale, IA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Albert; Henrik
Castle; Linda A.
Heckert; Matthew
Lu; Jian
Siehl; Daniel L.
Tao; Yumin |
Alameda
Mountain View
Union City
Union City
Menlo Park
Urbandale |
CA
CA
CA
IA
CA
IA |
US
US
US
US
US
US |
|
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC. (Johnston, IA)
|
Family
ID: |
45565770 |
Appl.
No.: |
13/208,960 |
Filed: |
August 12, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120042412 A1 |
Feb 16, 2012 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61401456 |
Aug 13, 2010 |
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61393507 |
Oct 15, 2010 |
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61501042 |
Jun 24, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/8274 (20130101); C12N 9/0069 (20130101); C12N
15/82 (20130101); C12N 15/8221 (20130101); C12Y
113/11027 (20130101) |
Current International
Class: |
C12N
15/82 (20060101); C12N 9/02 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 97/49816 |
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Dec 1997 |
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WO |
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WO 0032757 |
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Jun 2000 |
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WO |
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WO 0220741 |
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Mar 2002 |
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WO |
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WO 02/46387 |
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Jun 2002 |
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WO |
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WO 2009/144079 |
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Dec 2009 |
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WO |
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WO 2010/085705 |
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Jul 2010 |
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WO |
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Other References
Lee et al. (Plant Physiology, (2006), vol. 140: pp. 466-483). cited
by examiner .
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cited by examiner .
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French, English translation provided). cited by examiner .
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cited by examiner .
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1005-1016). cited by examiner .
WO 0220741 A1 english translation. cited by examiner .
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Proteins Based on Their N-terminal Amino Acid Sequence," J.Mol.
Biol., 2000, pp. 1005-1016, vol. 300, Academic Press, USA. cited by
applicant .
Fritze, I., et al., "The Crystal Structures of Zea mays and
Arabidopsis 4-Hydroxyphenylpyruvate Dioxygenase," Plant Physiology,
Apr. 2004, pp. 1388-1400, vol. 134, American Society of Plant
Biologists, USA. cited by applicant .
Garcia, I., et al., "Subcellular localization and purification of a
p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and
characterization of the corresponding cDNA," Biochem. J., 1997, pp.
761-769, vol. 325, Great Britain. cited by applicant .
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from Arabidopsis in Transgenic Tobacco," Plant Physiology, Apr.
1999, pp. 1507-1516, vol. 119, American Society of Plant
Physiologists, USA. cited by applicant .
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localization using N-terminal targeting sequences, sequence motifs
and amino acid composition," Bioinformatics, 2006, pp. 1158-1165,
vol. 22(10), Oxford University Press. cited by applicant .
Yang, C., et al., "Structural Basis for Herbicidal Inhibitor
Selectivity Revealed by Comparison of Crystal Structures of Plant
and Mammalian 4-Hydroxyphenylpyruvate Dioxygenases," Biochemistry,
2004, pp. 10414-10423, vol. 43, American Chemical Society, USA.
cited by applicant .
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cited by applicant .
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cited by applicant .
Castle, L., et al, "Discovery and Directed Evolution of a
Glyphosate Tolerance Gene," Science, 2004, vol. 304(5674), pp.
1151-1154. cited by applicant .
Database EMBL--Accession No. AF251071, "Oryza sativa seed protein
B32E mRNA, partial cds," 2002, pp. 1-2. cited by applicant .
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biosynthesis to tocopherol and plastoquinone in spinach
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applicant.
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Primary Examiner: Fox; David T
Assistant Examiner: Shapiro; Jared
Attorney, Agent or Firm: Ballard Spahr LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
Ser. No. 61/401,456, filed Aug. 13, 2010; U.S. Provisional Ser. No.
61/393,507, filed Oct. 15, 2010; and, U.S. Provisional Ser. No.
61/501,042, filed Jun. 24, 2011; each of which is herein
incorporated by reference.
Claims
That which is claimed:
1. A chimeric polynucleotide comprising a nucleotide sequence
encoding a chloroplast transit peptide operably linked to a
heterologous polynucleotide encoding a polypeptide of interest,
wherein the polypeptide of interest confers herbicide resistance,
wherein said chloroplast transit peptide comprises an amino acid
sequence having at least 90% sequence identity to SEQ ID NO: 58 and
having at least 17 consecutive amino acids of SEQ ID NO: 58,
wherein the 17 consecutive amino acids are from amino acids 1 to
41.
2. The chimeric polynucleotide of claim 1, wherein said chloroplast
transit peptide comprises SEQ ID NO: 58.
3. The chimeric polynucleotide of claim 1, wherein said polypeptide
of interest comprises a 4-hydroxphenylpyruvate dioxygenase (HPPD)
polypeptide having HPPD activity.
4. A nucleic acid construct comprising the chimeric polynucleotide
of claim 1.
5. The nucleic acid construct of claim 4, further comprising a
promoter operably linked to said chimeric polynucleotide.
6. A cell comprising at least one chimeric polynucleotide of claim
1.
7. The cell of claim 6, wherein said cell is a plant cell.
8. The cell of claim 7, wherein said polynucleotide or nucleic acid
construct is stably incorporated into the genome of said plant
cell.
9. The cell of claim 7, wherein said plant cell is from a
monocot.
10. The cell of claim 9, wherein said monocot is maize, wheat,
rice, barley, sorghum, or rye.
11. The cell of claim 7, wherein said plant cell is from a
dicot.
12. The cell of claim 11, wherein the dicot is soybean, Brassica,
sunflower, cotton, or alfalfa.
13. A plant comprising at least one plant cell of claim 7.
14. A plant explant comprising at least one plant cell of claim
7.
15. A transgenic seed produced by the plant of claim 13, wherein
said seed comprises said chimeric polynucleotide.
16. The plant cell of claim 7, wherein the plant cell further
comprises at least one polypeptide imparting tolerance to a
herbicide.
17. The plant cell of claim 16, wherein said at least one
polypeptide imparting tolerance to a herbicide comprises: (a) a
sulfonylurea-tolerant acetolactate synthase; (b) an
imidazolinone-tolerant acetolactate synthase; (c) a
glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase;
(d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a
glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl
transferase; (g) a protoporphyrinogen oxidase; (h) an auxin enzyme
or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A
carboxylase (ACCase).
18. A chimeric polypeptide encoded by the polynucleotide of claim
1.
19. A method of targeting a polypeptide of interest to a
chloroplast comprising expressing a chimeric polynucleotide of
claim 1 or the nucleic acid construct of claim 4 in a plant
cell.
20. A method of targeting a polypeptide of interest to a
chloroplast, comprising: introducing the chimeric polynucleotide of
claim 1 or a nucleic acid construct comprising said chimeric
polynucleotide in a plant cell and expressing said chimeric
polynucleotide in the plant cell.
21. The method of claim 19, wherein said method further comprises
regenerating a transgenic plant from said plant cell.
22. The method of claim 19, wherein said plant cell is from a
dicot.
23. The method of claim 22, wherein said dicot is selected from the
group consisting of soybean, Brassica, sunflower, cotton, and
alfalfa.
24. The method of claim 19, wherein said plant cell is from a
monocot.
25. The method of claim 24, wherein said dicot is selected from the
group consisting of maize, wheat, rice, barley, sorghum, and
rye.
26. The method of claim 19, wherein the plant cell further
comprises at least one polypeptide imparting tolerance to a
herbicide.
27. The method of claim 26, wherein said at least one polypeptide
imparting tolerance to a herbicide comprises: (a) a
sulfonylurea-tolerant acetolactate synthase; (b) an
imidazolinone-tolerant acetolactate synthase; (c) a
glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase;
(d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a
glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl
transferase; (g) a protoporphyrinogen oxidase; (h) an auxin enzyme
or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A
carboxylase (ACCase).
28. An expression cassette comprising a nucleic acid molecule
operably linked to a heterologous promoter, wherein said
heterologous promoter drives expression in a plant and wherein said
nucleic acid molecule is selected from the group consisting of: a)
a nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO: 60; b) a nucleic acid molecule that encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO: 57; c) a nucleic
acid molecule that encodes a polypeptide comprising an amino acid
sequence having at least 90% sequence identity to the amino acid
sequence of SEQ ID NO: 57, wherein said nucleotide sequence encodes
a polypeptide that has HPPD activity and is transported into the
chloroplast, wherein a polypeptide comprising an amino acid
sequence having at least 90% sequence identity to the amino acid
sequence of SEQ ID NO: 57 comprises 17 consecutive amino acids of
SEQ ID NO:58, wherein the 17 consecutive amino acids are from amino
acids 1 to 41; and, d) a full length complement of any of
a)-c).
29. A plant cell comprising at least one expression cassette of
claim 28.
30. The plant cell of claim 29, wherein said plant cell is a
monocot.
31. The plant cell of claim 30, wherein said monocot is maize,
wheat, rice, barley, sorghum, or rye.
32. The plant cell of claim 29, wherein said plant is from a
dicot.
33. The plant cell of claim 32, wherein said dicot is soybean,
Brassica, sunflower, cotton, or alfalfa.
34. A plant comprising at least one plant cell of claim 29.
35. A transgenic seed produced by the plant of claim 34, wherein
the seed comprises said expression cassette.
36. The plant cell of claim 29, wherein the plant cell further
comprises at least one polypeptide imparting tolerance to an
additional herbicide.
37. The plant cell of claim 36, wherein said at least one
polypeptide imparting tolerance to an additional herbicide
comprises: (a) a sulfonylurea-tolerant acetolactate synthase; (b)
an imidazolinone-tolerant acetolactate synthase; (c) a
glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase;
(d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a
glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl
transferase; (g) a protoporphyrinogen oxidase; (h) an auxin enzyme
or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A
carboxylase (ACCase).
38. The plant cell of claim 36, wherein said at least one
polypeptide imparting tolerance to an additional herbicide
comprises a high resistance allele of acetolactate synthase, a
glyphosate-N-acetyltransferase polypeptide, or both.
Description
FIELD OF THE INVENTION
This invention is in the field of molecular biology. More
specifically, this invention pertains to targeting sequences of
interest to a chloroplast by employing a novel chloroplast transit
peptide.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named 408355seqlist.txt, created on Aug. 12, 2011, and
having a size of 108 KB and is filed concurrently with the
specification. The sequence listing contained in this ASCII
formatted document is part of the specification and is herein
incorporated by reference in its entirety
BACKGROUND OF THE INVENTION
Plastids are a heterogeneous family of organelles found
ubiquitously in plants and algal cells. Most prominent are the
chloroplasts, which carry out such essential processes as
photosynthesis and the biosynthesis of fatty acids as well as of
amino acids. Chloroplasts are complex organelles composed of six
distinct suborganellar compartments: three different membranes (the
two envelope membranes and the internal thylakoid membranes) and
three compartments (the innermembrane space of the envelope, the
stroma and the thylakoid lumen.) More than 98% of all plastid
proteins are translated on cytosolic ribosomes. Such proteins are
posttranslationally targeted to and imported into the organelle.
For a review, see, Jarvis et al. (2008) New Phytologist
179:257-285. Such translocation is mediated by multiprotein
complexes in the outer and inner envelope membranes called TOC
(Translocon at the Outer envelope membrane of Chloroplasts) and TIC
(Translocon at the Inner envelope membrane of Chloroplasts). See,
Soll et al. (2004) Nature Reviews. Molecular Cell Biology
5:198-208, Bedard et al. (2005) Journal of Experimental Botany
56:2287-2320, Kessler et al. (2006) Traffic 7:248-257, and Smith et
al. (2006) Canadian Journal of Botany 84:531-542. Once the
chloroplast precursor enters the stroma, the transit peptide if
cleaved off, leaving the remaining part of the protein to take on
its final confirmation or engage one of a number of different
sorting pathways. See, Keegstra et al. (1999) Plant Cell
11:557-570, Jarvis et al. (2004) and Gutensohn et al. (2006)
Journal of Plant Physiology 163:333-347.
Methods and compositions are needed to allow heterologous
polypeptides to be targeted to the chloroplast.
BRIEF SUMMARY OF THE INVENTION
Chimeric polynucleotides comprising a nucleotide sequence encoding
a chloroplast transit peptide operably linked to a heterologous
polynucleotide of interest are provided, wherein the chloroplast
transit peptide comprises an amino acid sequence having the
consensus monocot HPPD chloroplast transit peptide sequence as set
forth in SEQ ID NO:1 or a biologically active variant or fragment
thereof or wherein the chloroplast transit peptide comprises the
sequence as set forth in SEQ ID NO: 58 or a biologically active
variant or fragment thereof. Chimeric polypeptides encoding the
same, as well as, cells, plant cells, plants and seeds are further
provided which comprise the chimeric polynucleotides. Methods of
use of the various sequences are also provided.
Compositions further include novel HPPD polypeptides and
polynucleotides encoding the same as set forth in SEQ ID NOS: 57
and 60 or active variants and fragments thereof. Such sequences
comprise the chloroplast transit peptide as set forth in SEQ ID NO:
58 or an active variant or fragment thereof. Cells, plant cells,
plants and seeds are further provided which comprise such
sequences. Methods of use of the various sequences are also
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides an amino acid alignment of HPPD from various
monocot plants. HPPD from Hordeum vulgare is set forth in SEQ ID
NO: 11. HPPD from Avena sativa is set forth in SEQ ID NO: 12. HPPD
from Oryza sativa is set forth in SEQ ID NO:13. HPPD from Triticum
aestivum is set forth in SEQ ID NO: 14. HPPD from Zea mays is set
forth in SEQ ID NO: 10. HPPD from Sorghum bicolor is set forth in
SEQ ID NO: 54. The underlining denotes amino acid residues sharing
identity and the shading further displays the conserved amino acid
residues.
FIG. 2 provides an amino acid alignment of HPPD polypeptides from
various dicot plants compared to Zea mays SEQ ID NO: 10. HPPD from
Daucus carota is set forth in SEQ ID NO: 15. HPPD from Solenostemon
scutellarioides is set for in SEQ ID NO: 16. HPPD from Picea
sitchenis is set for in SEQ ID NO: 17. HPPD from Abutilon
theophrasti is set forth in SEQ ID NO: 18. HPPD from Arabidopsis
thaliana is set forth in SEQ ID NO: 19. The HPPD from Brassica rapa
is set forth in SEQ ID NO: 20. HPPD from Coptis japonica is set
forth in SEQ ID NO: 21. HPPD from Vitis vinifera is set forth in
SEQ ID NO: 22. HPPD from Glycine max is set forth in SEQ ID NO: 23.
HPPD from Medicago truncatula is set forth in SEQ ID NO: 24.
FIG. 3 provides an alignment showing the diversity found in the
N-terminal amino acids of HPPD polypeptides from moncot plants,
dicot plants, microbes, a green alga and mammals.
FIG. 4A provides an alignment of the N-terminal amino acids of the
HPPD polypeptide from various monocot plants. Amino acids 1-52 of
the Zea mays HPPD are set forth in SEQ ID NO:3; amino acids 1-52 of
the Sorghum bicolor HPPD are set forth in SEQ ID NO:4; amino acids
1-52 of the Oryza sativa HPPD are set forth in SEQ ID NO: 5; amino
acids 1-48 of the Triticum aestivum HPPD are set forth in SEQ ID
NO: 6; amino acids 1-46 of the Hordeum vulgare HPPD are set forth
in SEQ ID NO:7; amino acids 1-47 of the Avena sativa HPPD are set
forth in SEQ ID NO: 8; and the consensus sequence is set forth in
SEQ ID NO: 2. FIG. 4B provides the % identity shared between the
N-terminal regions of the HPPD polypeptides shown in FIG. 4A. The
alignment was generated using AlignX which uses a modified Clustal
W algorithm (program in Vector NTI (Invitrogen).)
FIG. 5A-C provides fluorescence microscopy of maize leaf tissue
transfected with chloroplast-targeted or untargeted DsRed. FIG. 5A
shows fluorescence observed in maize leaf transfected with
ZmRCA1-Pro::RCA1CTP-Ds-Red2, 1000.times.. FIG. 5B shows
fluorescence observed in maize leaf transfected with
ZmRCA1-Pro::N-term-ZmHPPD-Ds-Red2, 1000.times.. FIG. 5C shows
fluorescence observed in maize leaf transfected with untargeted
Ds-Red2, 1000.times.. Photos on the left were of the same sample
taken with white light.
FIG. 6 provides fluorescence microscopy of maize leaf tissue
transformed by co-bombardment with plasmids coding for cycle 3
green fluorescence protein in combination with a plasmid coding for
either Rubisco activase CTP fused to DsRed (A-D), the N-terminal 50
amino acids of maize HPPD fused to DsRed (E-G), or untargeted DsRed
(H-J). The red channel (Figs. B, F and I) shows the pattern of
DsRed fluorescence, the green channel (Figs. D, G, and J) cytosolic
C3GFP fluorescence and the blue channel (Fig. C) chlorophyll
autofluorescence. Overlays of the red and green channels are shown
in figures A, E and H.
FIG. 7A-E provides an alignment of additional HPPD sequences.
FIG. 8 shows transient expression of Gm HPPD-AcGFP fusion proteins
in soy leaf cells. Epifluorescence micrographs of soy leaf sections
infiltrated with both untargeted (cytoplasmic) DsRed2 and Gm-HPPD N
terminus fusions to AcGFP. A and C. With both vectors red
fluorescence is seen in the cytoplasm while plastids remain dark.
B. When AcGFP is fused to Gm-HPPD amino acids 42-86 (from SEQ ID
NO: 57), green fluorescence is seen in the cytoplasm and plastids
remain dark. D. When AcGFP is fused to Gm HPPD amino acids 1-86
(from SEQ ID NO: 57), green fluorescence is clearly seen in
plastids of infected cells.
FIG. 9 shows that 50 amino acids of the maize HPPD N-terminus
effectively targeted DsRed to plastids. N-terminal 0, 10, 20, 30,
40 or 50 amino acids of Zea Mays HPPD fused to Ds-Red. A-F: DsRed
fluorescence micrographs A) 0aa, B) 10aa, C) 20aa, D) 30aa, E) 40aa
F) 50aa.
FIG. 10 shows AcGFP fluorescence confocal micrograph of soybean
leaf epidermal cell transiently expressing AcGFP linked to 50 amino
acids of maize HPPD N-terminus in both the chloroplasts and
cytoplasm.
FIG. 11 shows a leaf section of stably transformed soybean leaf
showing subcellular localization of Z. mays HPPD protein. CP:
chloroplast; CY: cytosol; NUC: nucleus.
DETAILED DESCRIPTION OF THE INVENTION
The present inventions now will be described more fully hereinafter
with reference to the accompanying drawings, in which some, but not
all embodiments of the inventions are shown. Indeed, these
inventions may be embodied in many different forms and should not
be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
Many modifications and other embodiments of the inventions set
forth herein will come to mind to one skilled in the art to which
these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
I. Overview
In the production of transgenic plants it is often useful to direct
foreign proteins to specific subcellular locations, e.g., the
plastid, vacuole, mitochondria, or ER. When the gene is translated,
the resulting protein has the transit peptide fused to the amino
terminus of the protein of interest, and thus the protein is
directed to the desired subcellular compartment. Of particular
interest is the identification of transit peptides that will direct
transport to a plastid. As used herein, a "plastid" refers to an
organelle present in plant cells that stores and manufactures
chemical compounds used by the cell, such as starch, fatty acids,
terpenes, and that has been derived from a proplastid. Thus,
plastids of plants typically have the same genetic content.
Plastids include chloroplasts, which are responsible for
photosynthesis, amyloplasts, chromoplasts, statoliths, leucoplasts,
elaioplasts, and proteinoplasts. Plastids contain photosynthetic
machinery and many additional biosynthetic enzymes including those
leading to the production of fatty acids, amino acids, carotenoids,
terpenoids, and starch. Thus, there is a need for the ability to
target polypeptides of interest to plastids to modulate or alter
the physiological processes that occur within these organelles. In
addition, some polypeptides are toxic when expressed recombinantly
in the cytoplasm. Because plastids are subcompartments, it is
possible to target polypeptides of interest to the plastids to
sequester them from the cytoplasm, and thus allow for higher
expression levels. Furthermore, expression of recombinant
polypeptides in plastids may facilitate isolation of the
polypeptide for various applications. As discussed in further
detail herein, novel CTP polypeptides from hydroxyphenylpyruvate
dioxygenase polypeptides are provided which can be used in plastid
targeting.
PSORT, a program that uses sequence data to predict organelle
targeting, does not identify the N-terminal region of plant
hydroxyphenylpyruvate dioxygenase (HPPD) proteins as a plastid
targeting polypeptide. However, as demonstrated herein, HPPD
polypeptides do contain a plastid targeting sequence which can be
employed in a variety of methods and compositions to aid in
targeting polypeptides of interest to the plastids. Thus,
compositions and methods are provided for the targeting of
polypeptides of interest to the chloroplast of a plant or plant
cell.
The compositions provided herein include polynucleotides comprising
a nucleotide sequence encoding a chloroplast transit peptide (CTP)
derived from an HPPD polypeptide operably linked to a nucleotide
sequence encoding a polypeptide of interest. The CTP-encoding
sequences disclosed herein, when assembled within a DNA construct
such that the CTP-encoding sequence is operably linked to a
nucleotide sequence encoding the polypeptide of interest,
facilitate co-translational or post-translational transport of the
peptide of interest to the chloroplast of a plant cell.
II. Chloroplast Transit Peptides
Chloroplasts are organelles found in plant cells and eukaryotic
algae that conduct photosynthesis. The chloroplast is a complex
cellular organelle composed of three membranes: the inner envelope
membrane, the outer envelope membrane, and the thylakoid membrane.
The membranes together enclose three aqueous compartments termed
the intermediate space, the stroma, and the thylakoid lumen. While
chloroplasts contain their own circular genome, many constituent
chloroplast proteins are encoded by the nuclear genes and are
cytoplasmically-synthesized as precursor forms which contain
N-terminal extensions known as chloroplast transit peptides (CTPs).
As used herein, the term "chloroplast transit peptide" or "CTP"
refers to the N-terminal portion of a chloroplast precursor protein
and is instrumental for specific recognition of the chloroplast
surface and in mediating the post-translational translocation of
pre-proteins across the chloroplast envelope and into the various
subcompartments within the chloroplast (e.g. stroma, thylakoid and
thylakoid membrane). Thus, as used herein, a polypeptide having
"CTP activity" comprises a polypeptide which when operably linked
to the N-terminal region of a protein of interest facilitates
translocation of the polypeptide of interest to the
chloroplast.
In one embodiment, a CTP is provided comprising the following HPPD
CTP consensus sequence.
TABLE-US-00001 (SEQ ID NO: 1) MPPTP(T/A) (T/P/A) (T/P/A) (A/T)
(G/T/A) (G/T/A) (G/A/*) (A/*) (G/V/*) (A/S/V) AA(A/S) (A/S/V) (T/A)
(P/G/*)E(HN/Q) A(A/G/R) (F/P/R) (R/*)(L/*)(V/*)(G/S/*)
(H/F/*)(R/H/P) (R/N)(F/M/V) VR(F/A/V) NPRSDRF
(H/Q/P)(T/A/V)L(A/S)FHHVE
or an active variant or fragment thereof, where the * indicates
that that amino acid position is not represented (ie. a gap in the
alignment).
In further embodiments, a synthetic consensus HPPD sequence
comprising a CTP is provided having the following sequence:
TABLE-US-00002 (SEQ ID NO: 2)
MPPTPTTAAATGAGAAAAVTPEHAAFRLVGHRRFVRFNPRSDRFH TLAFHHVE
or an active variant or fragment thereof.
In still other embodiments, a CTP is provided that comprises the
N-terminal region of any HPPD polypeptide, including for example,
the N-terminal region of a monocot HPPD polypeptide or a dicot
HPPD. In one embodiment, the CTP can comprise amino acids 1-53,
1-17, 1-19, 1-20, 1-23, 1-30, 1-40 and 1-60 or a variant or
fragment thereof of any monocot HPPD polypeptide. For example, the
CTP can comprise any one of SEQ ID NO:3 (amino acids 1-52 of the
Zea mays HPPD); SEQ ID NO: 4 (amino acids 1-52 of the Sorghum
bicolor HPPD); SEQ ID NO: 5 (amino acids 1-52 of the Oryza sativa
HPPD); SEQ ID NO: 6 (amino acids 1-48 of the Triticum aestivum
HPPD); SEQ ID NO:7 (amino acids 1-46 of the Hordeum vulgare HPPD);
SEQ ID NO:8 (amino acids 1-47 of the Avena sativa HPPD); or an
active variant or fragment of any one of SEQ ID NOS: 2, 3, 4, 5, 6,
7 or 8. The CTP-encoding sequence can further comprise any
N-terminal region (about amino acids 1-53, 1-17, 1-19, 1-20, 1-30,
1-40 and 1-60 or 1-23) of any of the HPPD polypeptides as set forth
in FIG. 2 or 7 or an active variant or fragment of such
polypeptides. In addition, the CTP can comprise the sequence of SEQ
ID NO:58 (amino acids 1-86 of the Soybean HPPD) or an active
variant or fragment thereof.
It is recognized that the various CTPs disclosed herein can be
modified to improve and/or alter the translocation of the
polypeptide of interest into the chloroplast. For example, the CTP
can contain additional regions that alter or improve the
interactions with cytosolic factors that facilitate the passage of
precursors from the ribosomes to the chloroplast surface. See, for
example, Hiltbrunner et al. (2001) Journal of Cell Biology
154:309-316, Jackson-Constan et al. (2001) Biochimica et Biophysica
Acta 1541:102-113, both of which are herein incorporated by
reference. Other regions can be employed to increase the efficiency
of chloroplast import. See, for example, May et al. (2000) Plant
Cell 12:53-64, Qbadou et al. (2006) EMBO Journal 25:1837-1837 and
Sohrt et al. (2000) Journal of Cell Biology 148:1213-1221, herein
incorporated by reference. Such regions may be native (derived from
a region of the HPPD polypeptide) or heterologous to the operably
linked HPPD CTP.
The various CTP disclosed herein can further comprise additional
sequences which modulate the final location of the polypeptide of
interest in the chloroplast. For example, the various CTPs
disclosed herein could further comprise a thylakoid lumen targeting
domain. Proteins to be targeted to the thylakoid lumen bear an
additional cleavable targeting signal, which like the transit
peptide, is removed once translocation is complete. The luminal
targeting peptides are extremely similar to the signal peptides
that mediate inner membrane transport in bacteria. See, for
example, Keegstra et al. (1999) Plant Cell 11:557-570, Jarvis
(2004) Current Biology 14: R1064-R1077, Gutensohn et al. (2006)
Journal of Plant Physiology 163:333-347, and Jarvis (2008) New
Phytologist 179:257-285, all of which are incorporated by reference
in their entirety, which discuss the various sorting pathways in a
chloroplast. Such regions which modulate the location of the
polypeptide of interest in a chloroplast may be native (derived
from a region of the HPPD polypeptide) or heterologous to the
operably linked HPPD CTP.
The term "chloroplast transit peptide cleavage site" refers to a
site between two amino acids in a chloroplast-targeting sequence at
which the chloroplast processing protease acts. CTPs target the
desired protein to the chloroplast and can facilitate the protein's
translocation into the organelle. This is accompanied by the
cleavage of the transit peptide from the mature polypeptide or
protein at the appropriate transit peptide cleavage site by a
chloroplast processing protease. Accordingly, a CTP further
comprises a suitable cleavage site for the correct processing of
the pre-protein to the mature polypeptide contained within the
chloroplast. In one non-limiting example, the CTP cleavage site is
after amino acid 23, between Q and A, in SEQ ID NO:1 which would
equate to the H/N/Q-A in SEQ ID NO: 1. As discussed above, the
sequences beyond the cleaved fragments may be important for
localization/transport efficiency and be employed with any of the
CTPs disclosed herein.
The term "chimeric" sequence refers to a sequence having two or
more heterologous sequences linked together. As used herein, a
"heterologous" CTP comprises a transit peptide sequence which is
foreign to the polypeptide of interest it is operably linked to. In
one embodiment, the heterologous chloroplast transit peptide
comprises any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 58 or an
active variant or fragment thereof.
Assays to determine the efficiency by which the CTP sequences of
the invention target a protein of interest to a chloroplast are
known. See, for example, Mishkind et al. (1985) J of Cell Biol
100:226-234, which is herein incorporated by reference in its
entirety. A reporter gene such as glucuronidase (GUS),
chloramphenicol acetyl transferase (CAT), or green fluorescent
protein (GFP) is operably linked to the CTP sequence. This fusion
is placed behind the control of a suitable promoter, ligated into a
transformation vector, and transformed into a plant or plant cell.
Following an adequate period of time for expression and
localization into the chloroplast, the chloroplast fraction is
extracted and reporter activity assayed. The ability of the
isolated sequences to target and deliver the reporter protein to
the chloroplast can be compared to other known CTP sequences. See,
de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769-780.
Protein import can also be verified in vitro through the addition
of proteases to the isolated chloroplast fraction. Proteins which
were successfully imported into the chloroplast are resistant to
the externally added proteases whereas proteins that remain in the
cytosol are susceptible to digestion. Protein import can also be
verified by the presence of functional protein in the chloroplast
using standard molecular techniques for detection, by evaluating
the phenotype resulting from expression of a chloroplast targeted
protein, or by microscopy.
As used herein, an "isolated" or "purified" polynucleotide or
polypeptide, or biologically active portion thereof, is
substantially or essentially free from components that normally
accompany or interact with the polynucleotide or polypeptide as
found in its naturally occurring environment. Thus, an isolated or
purified polynucleotide or polypeptide is substantially free of
other cellular material or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
Optimally, an "isolated" polynucleotide is free of sequences
(optimally protein encoding sequences) that naturally flank the
polynucleotide (i.e., sequences located at the 5' and 3' ends of
the polynucleotide) in the genomic DNA of the organism from which
the polynucleotide is derived. For example, in various embodiments,
the isolated polynucleotide can contain less than about 5 kb, 4 kb,
3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that
naturally flank the polynucleotide in genomic DNA of the cell from
which the polynucleotide is derived. A polypeptide that is
substantially free of cellular material includes preparations of
polypeptides having less than about 30%, 20%, 10%, 5%, or 1% (by
dry weight) of contaminating protein. When the polypeptide of the
invention or biologically active portion thereof is recombinantly
produced, optimally culture medium represents less than about 30%,
20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals.
As used herein, polynucleotide or polypeptide is "recombinant" when
it is artificial or engineered, or derived from an artificial or
engineered protein or nucleic acid. For example, a polynucleotide
that is inserted into a vector or any other heterologous location,
e.g., in a genome of a recombinant organism, such that it is not
associated with nucleotide sequences that normally flank the
polynucleotide as it is found in nature is a recombinant
polynucleotide. A polypeptide expressed in vitro or in vivo from a
recombinant polynucleotide is an example of a recombinant
polypeptide. Likewise, a polynucleotide sequence that does not
appear in nature, for example, a variant of a naturally occurring
gene is recombinant.
a. Polynucleotide and Polypeptide Fragments and Variants of
CTPs
Fragments and variants of the CTP-sequences are also encompassed by
the present invention. By "fragment" is intended a portion of the
polynucleotide or a portion of the amino acid sequence and hence
protein encoded thereby. Fragments of a polynucleotide may encode
protein fragments that retain CTP activity and are thus capable of
facilitating the translocation of a polypeptide of interest into
the chloroplast of a plant. Alternatively, fragments of a
polynucleotide that is useful as a hybridization probe generally do
not encode fragment proteins retaining biological activity. Thus,
fragments of a nucleotide sequence may range from at least about
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170 nucleotides or up to the full length CTP.
A fragment of polynucleotide that encodes a biologically active
portion of a CTP-polypeptide will encode at least 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 60, 65, 70, 75, 80, 85 contiguous amino acids,
or up to the total number of amino acids present in any one of SEQ
ID NOS: 1, 2, 3, 4, 5, 6, 7, 8 or 58 or any one of the N-terminal
regions (about amino acids 1-17, 1-19, 1-20, 1-23, 1-30, 1-40 or
about 1-53, 1-86) of the HPPD polypeptide as set forth in FIG. 2 or
7 or in any one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8 or 58.
Fragments of a CTP-encoding sequence that are useful as
hybridization probes or PCR primers generally need not encode a
biologically active portion of an HPPD protein.
"Variant" protein is intended to mean a protein derived from the
protein by deletion (i.e., truncation at the 5' and/or 3' end)
and/or a deletion or addition of one or more amino acids at one or
more internal sites in the native protein and/or substitution of
one or more amino acids at one or more sites in the native protein.
Variant proteins encompassed are biologically active, that is they
continue to possess the desired biological activity of the native
protein, that is, have CTP activity. Such variants may result from,
for example, genetic polymorphism or from human manipulation.
For polynucleotides, a variant comprises a polynucleotide having a
deletion (i.e., truncations) at the 5' and/or 3' end and/or a
deletion and/or addition of one or more nucleotides at one or more
internal sites within the native polynucleotide and/or a
substitution of one or more nucleotides at one or more sites in the
native polynucleotide. As used herein, a "native" polynucleotide or
polypeptide comprises a naturally occurring nucleotide sequence or
amino acid sequence, respectively. For polynucleotides,
conservative variants include those sequences that, because of the
degeneracy of the genetic code, encode the amino acid sequence of
one of the CTPs disclosed herein. Naturally occurring variants such
as these can be identified with the use of well-known molecular
biology techniques, as, for example, with polymerase chain reaction
(PCR) and hybridization techniques as outlined below. Variant
polynucleotides also include synthetically derived polynucleotides,
such as those generated, for example, by using site-directed
mutagenesis or gene synthesis but which still encode a CTP.
Biologically active variants of a CTP (and the polynucleotide
encoding the same) will have at least about 60%, 65%, 70%, 75%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the
polypeptide of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 58
or to the N-terminal region (about 1-53, about 1-17, about 1-19,
about 1-20, about 1-23, about 1-30, about 1-40, about 1-60, about
1-70, about 1-75, about 1-80, about 1-85) of the HPPD polypeptides
as set forth in FIGS. 2 and 6 or in any one of SEQ ID NOS:1-8 or
58.
The CTP-sequences and the active variants and fragments thereof may
be altered in various ways including amino acid substitutions,
deletions, truncations, and insertions. Methods for such
manipulations are generally known in the art. For example, amino
acid sequence variants and fragments of the CTPs can be prepared by
mutations in the DNA. Methods for mutagenesis and polynucleotide
alterations are well known in the art. See, for example, Kunkel
(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)
Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker
and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan
Publishing Company, New York) and the references cited therein.
Guidance as to appropriate amino acid substitutions that do not
affect biological activity of the protein of interest may be found
in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and
Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein
incorporated by reference. Conservative substitutions, such as
exchanging one amino acid with another having similar properties,
may be optimal.
Obviously, the mutations that will be made in the DNA encoding the
variant must not place the sequence out of reading frame and
optimally will not create complementary regions that could produce
secondary mRNA structure. See, EP Patent Application Publication
No. 75,444.
Variant polynucleotides and proteins also encompass sequences and
proteins derived from a mutagenic and recombinogenic procedure such
as DNA shuffling. With such a procedure, one or more different
CTP-sequences can be manipulated to create a new CTP possessing the
desired properties. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in
vivo. For example, using this approach, sequence motifs encoding a
domain of interest may be shuffled between the CTP sequences
disclosed herein and other known CTPs to obtain a new
polynucleotide coding for a polypeptide with an improved property
of interest, such as an improved efficiency of transport to the
chloroplast. Strategies for such DNA shuffling are known in the
art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA
91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al.
(1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.
Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Pat. Nos. 5,605,793 and 5,837,458.
III. Sequence Comparisons
The following terms are used to describe the sequence relationships
between two or more polynucleotides or polypeptides: (a) "reference
sequence", (b) "comparison window", (c) "sequence identity", and,
(d) "percent sequence identity."
(a) As used herein, "reference sequence" is a defined sequence used
as a basis for sequence comparison. A reference sequence may be a
subset or the entirety of a specified sequence; for example, as a
segment of a full-length cDNA or gene sequence, or the complete
cDNA or gene sequence or protein sequence.
(b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polypeptide sequence, wherein
the polypeptide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two polypeptides. Generally, the
comparison window is at least 5, 10, 15, or 20 contiguous amino
acids in length, or it can be 30, 40, 50, 100, or longer. Those of
skill in the art understand that to avoid a high similarity to a
reference sequence due to inclusion of gaps in the polypeptide
sequence a gap penalty is typically introduced and is subtracted
from the number of matches.
Methods of alignment of sequences for comparison are well known in
the art. Thus, the determination of percent sequence identity
between any two sequences can be accomplished using a mathematical
algorithm. Non-limiting examples of such mathematical algorithms
are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the
local alignment algorithm of Smith et al. (1981) Adv. Appl. Math.
2:482; the global alignment algorithm of Needleman and Wunsch
(1970) J. Mol. Biol. 48:443-453; the search-for-local alignment
method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.
85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc.
Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
Computer implementations of these mathematical algorithms can be
utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins et al. (1988) Gene 73:237-244
(1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.
(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS
8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.
The ALIGN program is based on the algorithm of Myers and Miller
(1988) supra. A PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4 can be used with the ALIGN program
when comparing amino acid sequences. The BLAST programs of Altschul
et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the invention. BLAST protein searches can be
performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the invention. BLASTP protein searches can be performed using
default parameters. See, blast.ncbi.nlm.nih.gov/Blast.cgi.
To obtain gapped alignments for comparison purposes, Gapped BLAST
(in BLAST 2.0) can be utilized as described in Altschul et al.
(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in
BLAST 2.0) can be used to perform an iterated search that detects
distant relationships between molecules. See Altschul et al. (1997)
supra. When utilizing BLAST, Gapped BLAST, or PSI-BLAST, the
default parameters of the respective programs (e.g., BLASTN for
nucleotide sequences, BLASTP for proteins) can be used. See
www.ncbi.nlm.nih.gov. Alignment may also be performed manually by
inspection.
In one embodiment, sequence identity/similarity values provided
herein refer to the value obtained using GAP Version 10 using the
following parameters: % identity and % similarity for an amino acid
sequence using GAP Weight of 8 and Length Weight of 2, and the
BLOSUM62 scoring matrix; or any equivalent program thereof. By
"equivalent program" is intended any sequence comparison program
that, for any two sequences in question, generates an alignment
having identical nucleotide or amino acid residue matches and an
identical percent sequence identity when compared to the
corresponding alignment generated by GAP Version 10.
GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.
48:443-453, to find the alignment of two complete sequences that
maximizes the number of matches and minimizes the number of gaps.
GAP considers all possible alignments and gap positions and creates
the alignment with the largest number of matched bases and the
fewest gaps. It allows for the provision of a gap creation penalty
and a gap extension penalty in units of matched bases. GAP must
make a profit of gap creation penalty number of matches for each
gap it inserts. If a gap extension penalty greater than zero is
chosen, GAP must, in addition, make a profit for each gap inserted
of the length of the gap times the gap extension penalty. Default
gap creation penalty values and gap extension penalty values in
Version 10 of the GCG Wisconsin Genetics Software Package for
protein sequences are 8 and 2, respectively. For nucleotide
sequences the default gap creation penalty is 50 while the default
gap extension penalty is 3. The gap creation and gap extension
penalties can be expressed as an integer selected from the group of
integers consisting of from 0 to 200. Thus, for example, the gap
creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or
greater.
GAP presents one member of the family of best alignments. There may
be many members of this family, but no other member has a better
quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the GCG Wisconsin Genetics Software Package is
BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci.
USA 89:10915).
(c) As used herein, "sequence identity" or "identity" in the
context of two polynucleotides or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity). When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the percent
sequence identity. Thus, for example, where an identical amino acid
is given a score of 1 and a non-conservative substitution is given
a score of zero, a conservative substitution is given a score
between zero and 1. The scoring of conservative substitutions is
calculated, e.g., as implemented in the program PC/GENE
(Intelligenetics, Mountain View, Calif.).
(d) As used herein, "percent sequence identity" means the value
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percent sequence
identity.
(e) Two sequences are "optimally aligned" when they are aligned for
similarity scoring using a defined amino acid substitution matrix
(e.g., BLOSUM62), gap existence penalty and gap extension penalty
so as to arrive at the highest score possible for that pair of
sequences. Amino acids substitution matrices and their use in
quantifying the similarity between two sequences are well-known in
the art and described, e.g., in Dayhoff et al. (1978) "A model of
evolutionary change in proteins." In "Atlas of Protein Sequence and
Structure," Vol. 5, Suppl. 3 (ed. M.O. Dayhoff), pp. 345-352. Natl.
Biomed. Res. Found., Washington, D.C. and Henikoff et al. (1992)
Proc. Natl. Acad. Sci. USA 89:10915-10919. The BLOSUM62 matrix is
often used as a default scoring substitution matrix in sequence
alignment protocols such as Gapped BLAST 2.0. The gap existence
penalty is imposed for the introduction of a single amino acid gap
in one of the aligned sequences, and the gap extension penalty is
imposed for each additional empty amino acid position inserted into
an already opened gap. The gap existence penalty is imposed for the
introduction of a single amino acid gap in one of the aligned
sequences, and the gap extension penalty is imposed for each
additional empty amino acid position inserted into an already
opened gap. The alignment is defined by the amino acids positions
of each sequence at which the alignment begins and ends, and
optionally by the insertion of a gap or multiple gaps in one or
both sequences, so as to arrive at the highest possible score.
While optimal alignment and scoring can be accomplished manually,
the process is facilitated by the use of a computer-implemented
alignment algorithm, e.g., gapped BLAST 2.0, described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402, and made available
to the public at the National Center for Biotechnology Information
Website (http://www.ncbi.nlm.nih.gov). Optimal alignments,
including multiple alignments, can be prepared using, e.g.,
PSI-BLAST, available through http://www.ncbi.nlm.nih.gov and
described by Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402.
As used herein, similarity score and bit score is determined
employing the BLAST alignment used the BLOSUM62 substitution
matrix, a gap existence penalty of 11, and a gap extension penalty
of 1. For the same pair of sequences, if there is a numerical
difference between the scores obtained when using one or the other
sequence as query sequences, a greater value of similarity score is
selected.
IV. Polynucleotides/Polypeptides of Interest
Any polynucleotide of interest (i.e., the "polypeptide of
interest") may be used with the CTP-encoding sequences disclosed
herein. Such polynucleotides/polypeptides of interest include, but
are not limited to, herbicide-tolerance coding sequences,
insecticidal coding sequences, nematicidal coding sequences,
antimicrobial coding sequences, antifungal coding sequences,
antiviral coding sequences, abiotic and biotic stress tolerance
coding sequences, or sequences modifying plant traits such as
yield, grain quality, nutrient content, starch quality and
quantity, nitrogen fixation and/or utilization, and oil content
and/or composition. More specific polynucleotides of interest for
the present invention include, but are not limited to, genes that
improve crop yield, polypeptides that improve desirability of
crops, genes encoding proteins conferring resistance to abiotic
stress, such as drought, nitrogen, temperature, salinity, toxic
metals or trace elements, or those conferring resistance to toxins
such as pesticides and herbicides, or to biotic stress, such as
attacks by fungi, viruses, bacteria, insects, and nematodes, and
development of diseases associated with these organisms. It is
recognized that any polypeptides of interest can be operably linked
to the CTP-encoding sequences of the invention and expressed in a
plant, so long as the polypeptide encoded by the polynucleotide is
functional in chloroplasts.
These nucleotide sequences of interest may encode proteins involved
in providing disease or pest resistance. By "disease resistance" or
"pest resistance" is intended that the plants avoid the harmful
symptoms that are the outcome of the plant-pathogen interactions.
Disease resistance and insect resistance genes such as lysozymes or
cecropins for antibacterial protection, or proteins such as
defensins, glucanases or chitinases for antifungal protection, or
Bacillus thuringiensis endotoxins, protease inhibitors,
collagenases, lectins, or glycosidases for controlling nematodes or
insects are all examples of useful gene products.
"Pest" includes, but is not limited to, insects, fungi, bacteria,
viruses, nematodes, mites, ticks, and the like. Insect pests
include insects selected from the orders Coleoptera, Diptera,
Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,
Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura,
Siphonaptera, Trichoptera, etc., particularly Coleoptera,
Lepidoptera, and Diptera. Viruses include but are not limited to
tobacco or cucumber mosaic virus, ringspot virus, necrosis virus,
maize dwarf mosaic virus, etc. Nematodes include but are not
limited to parasitic nematodes such as root knot, cyst, and lesion
nematodes, including Heterodera spp., Meloidogyne spp., and
Globodera spp.; particularly members of the cyst nematodes,
including, but not limited to, Heterodera glycines (soybean cyst
nematode); Heterodera schachtii (beet cyst nematode); Heterodera
avenae (cereal cyst nematode); and Globodera rostochiensis and
Globodera pailida (potato cyst nematodes). Lesion nematodes include
but are not limited to Pratylenchus spp. Fungal pests include those
that cause leaf, yellow, stripe and stem rusts.
An "herbicide resistance protein" or a protein resulting from
expression of an "herbicide resistance-encoding nucleic acid
molecule" includes proteins that confer upon a cell the ability to
tolerate a higher concentration of an herbicide than cells that do
not express the protein, or to tolerate a certain concentration of
an herbicide for a longer period of time than cells that do not
express the protein. Herbicide resistance traits may be introduced
into plants by genes coding for resistance to herbicides that act
to inhibit the action of acetolactate synthase (ALS), in particular
the sulfonylurea-type herbicides, genes coding for resistance to
herbicides that act to inhibit the action of glutamine synthase,
such as phosphinothricin or basta (e.g., the bar gene), glyphosate
(e.g., the EPSP synthase gene), HPPD inhibitors (e.g, the HPPD
gene) or other such genes known in the art. See, for example, U.S.
Pat. Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876,
7,169,970, and 6,867,293, each of which is herein incorporated by
reference.
Polynucleotides that improve crop yield include dwarfing genes,
such as Rht1 and Rht2 (Peng et al. (1999) Nature 400:256-261), and
those that increase plant growth, such as ammonium-inducible
glutamate dehydrogenase. Polynucleotides that improve desirability
of crops include, for example, those that allow plants to have a
reduced saturated fat content, those that boost the nutritional
value of plants, and those that increase grain protein.
Polynucleotides that improve salt tolerance are those that increase
or allow plant growth in an environment of higher salinity than the
native environment of the plant into which the salt-tolerant
gene(s) has been introduced.
Polynucleotides/polypeptides that influence amino acid biosynthesis
include, for example, anthranilate synthase (AS; EC 4.1.3.27) which
catalyzes the first reaction branching from the aromatic amino acid
pathway to the biosynthesis of tryptophan in plants, fungi, and
bacteria. In plants, the chemical processes for the biosynthesis of
tryptophan are compartmentalized in the chloroplast. See, for
example, US Pub. 20080050506, herein incorporated by reference.
Additional sequences of interest include Chorismate Pyruvate Lyase
(CPL) which refers to a gene encoding an enzyme which catalyzes the
conversion of chorismate to pyruvate and pHBA. The most well
characterized CPL gene has been isolated from E. coli and bears the
GenBank accession number M96268. See, U.S. Pat. No. 7,361,811,
herein incorporated by reference. Additional sequences of interest
are discussed in more detail below.
a. Hydroxyphenylpyruvate Dioxygenase (HPPD) Polynucleotides and
Polypeptides
In one embodiment, the CTP-encoding sequence is operably linked to
a heterologous polynucleotide encoding a hydroxphenylpyruvate
dioxygenase (HPPD) polypeptide. Various HPPD polypeptides and
active variants and fragments thereof are known, as discussed
below.
Hydroxyphenylpyruvate dioxygenase (HPPD) converts
hydroxyphenylpyruvate, derived from the aromatic amino acid
biosynthesis pathway, to homogentisate. In plants, homogentisate is
a precursor of tocopherols and plastoquinones, an electron carrier
essential in the biosynthesis of carotenoids. Consequently, when
HPPD is inhibited by herbicide inhibitors, the plant can not
protect itself from the radicals generated by light activation of
chlorophyll. More specifically, inhibition of HPPD polypeptide
leads to the depletion of protective pigments in the plant tissue
resulting in bleaching of tissues which leaves the plants
vulnerable to damage by light. HPPD inhibitors are an important
class of herbicides. Transgenes that confer crop tolerance to HPPD
inhibitors would be of significant value, especially for managing
weed resistance to glyphosate.
As used herein, "Hydroxyphenylpyruvate dioxygenase" and "HPPD"
"4-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase (4-HPPD)"
and "p-hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase
(p-OHPP)" are synonymous and refer to a non-heme iron-dependent
oxygenase that catalyzes the conversion of 4-hydroxyphenylpyruvate
to homogentisate. In organisms that degrade tyrosine, the reaction
catalyzed by HPPD is the second step in the pathway. In plants,
formation of homogentisate is necessary for the synthesis of
plastoquinone, an essential redox cofactor, and tocopherol. The
structures of various HPPD polypeptides are known. See, for
example, FIG. 1 which provides the phylogenetic diversity of
several monocot HPPD polypeptides, including sequences from Hordeum
vulgare, Avena sativa, Oryza sativa, Triticum aestivum, and Zea
mays. FIG. 2 provides the phylogenetic diversity of several dicot
HPPD polypeptides including Daucus carota, Solenosteman
sautellarioides, Picea sitchensis, Abutilon theophrasti,
Arabidopsis thaliana, Brassica rapa, Coptis japonica, Vitis
vinifera, Glycine max, and Medicago truncatula. HPPD polypeptides
from microbes and mammals are also known and non-limiting examples
of these sequences appear in FIG. 7.
Various variants of HPPD sequences are also known. See, for
example, U.S. Provisional Application 61/401,456, filed Aug. 13,
2010, Compositions and Methods Comprising Sequences having
Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity, herein
incorporated by reference in it entirety. See, also, US
2003/0066102, WO97/49816, US 2010/0197503, U.S. Pat. No. 7,312,379,
U.S. Pat. No. 6,768,044, U.S. Pat. No. 6,245,698, U.S. Pat. No.
6,268,549, and U.S. Pat. No. 6,118,050, the contents of each is
herein incorporated by reference in its entirety. A review of the
various structures of HPPD polypeptides from microbes, mammals and
plants can be found, for example, in Moran et al. (2005) Archives
of Biochemistry and Biophysics 433:117-128, herein incorporated by
reference in its entirety.
As used herein, "hydroxyphenylpyruvate dioxygenase activity" or
"HPPD activity" refers to the conversion of 4-hydroxyphenylpyruvate
to homogentisate. As used herein, a polypeptide having "HPPD
activity" comprises an HPPD polypeptide or an active variant or
fragment thereof that retains sufficient HPPD activity such that
(i) when expressed at sufficient levels in a cell that requires
HPPD activity for viability, the HPPD polypeptide or active variant
or fragment thereof exhibits sufficient HPPD activity to maintain
viability of the cell in which it is expressed; or (ii) when
expressed in a cell that requires HPPD activity for viability, the
HPPD polypeptide or active variant or fragment thereof, when
expressed in combination with one or more additional HPPD
polypeptides results in the viability of the cell. Methods to
determine such kinetic parameters (i.e., K.sub.m, k.sub.cat,
k.sub.cat/K.sub.m) are known. See, for example, U.S. Provisional
Application 61/401,456, filed Aug. 13, 2010 Compositions and
Methods Comprising Sequences having Hydroxyphenylpyruvate
Dioxygenase (HPPD) Activity, herein incorporated by reference.
In order to provide plants with tolerance to commercially useful
application rates of at least one desired HPPD inhibitor, it is
advantageous to use polynucleotides which encode HPPD polypeptides
having sufficient HPPD activity and having an insensitivity to
inhibition by at least one or more HPPD inhibitor.
As used herein, an "HPPD inhibitor" comprises any compound or
combinations of compounds which decrease the ability of HPPD to
catalyze the conversion of 4-hydroxyphenylpyruvate to
homogentisate. In specific embodiments, the HPPD inhibitor
comprises a herbicidal inhibitor of HPPD. Non-limiting examples of
HPPD inhibitors include, triketones (such as, mesotrione,
sulcotrione, topramezone, and tembotrione); isoxazoles (such as,
pyrasulfotole and isoxaflutole); pyrazoles (such as, benzofenap,
pyrazoxyfen, and pyrazolynate); and benzobicyclon. Agriculturally
acceptable salts of the various inhibitors include salts, the
cations or anions of which are known and accepted in the art for
the formation of salts for agricultural or horticultural use. See,
for example, WO2005/053407 herein incorporated by reference.
The insensitivity of an HPPD inhibitor can be determined by
assaying the insensitivity of a cell, a plant, a plant cell
expressing the HPPD polypeptide or active fragment or variant
thereof. In such instances, the cell, plant, or plant cell
expressing an HPPD sequence displays an insensitivity to an HPPD
inhibitor or to a combination of HPPD inhibitors when compared to a
control cell, plant or plant cell not expressing the HPPD sequence.
"Increased tolerance" to a herbicide is demonstrated when plants
which display the increased tolerance to a herbicide are subjected
to the HPPD inhibitor and a dose/response curve is shifted to the
right when compared with that provided by an appropriate control
plant. Such dose/response curves have "dose" plotted on the x-axis
and "percentage injury", "herbicidal effect" etc. plotted on the
y-axis. Plants which are substantially "resistant" or "tolerant" to
the herbicide exhibit few, if any, bleached, necrotic, lytic,
chlorotic or other lesions and are not stunted, wilted or deformed
when subjected to the herbicide at concentrations and rates which
are typically employed by the agricultural community to kill weeds
in the field.
V. Novel Hydroxyphenylpyruvate Dioxygenase (HPPD) Sequences
Compositions are further provided comprising a novel HPPD
polypeptide comprising the CTP set forth in SEQ ID NO: 58 and
active variants and fragments thereof. In specific embodiments,
such HPPD encoding sequences include the polynucleotide set forth
in SEQ ID NO: 60 and the polypeptide set forth in SEQ ID NO: 57,
and active variants and fragments thereof. Such polypeptides are
capable of being transported into the chloroplast of a plant cell.
In some embodiments, the polynucleotide set forth in SEQ ID NO: 60
or an active variant or fragment thereof is operably linked to a
heterologous promoter.
In specific embodiments, active fragments and variants of the HPPD
sequence as set forth in SEQ ID NO: 60 are provided. Such fragments
comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200,
1,300, or 1,450 contiguous nucleotides, or up to the number of
nucleotides present in SEQ ID NO: 60. Generally, variants of SEQ ID
NO: 60 will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more sequence identity to SEQ ID NO: 60 as determined by sequence
alignment programs and parameters described elsewhere herein.
Active fragments and variants of SEQ ID NO: 60 will continue to
encode a polypeptide having HPPD activity and which can be
transported into the chloroplast of a plant cell.
The HPPD promoter as described in SEQ ID NO:1 of U.S. Provisional
Application No. 61/501,042 leads to the production of at least two
major transcripts from at least two transcription start sites (TSS1
and TSS2, see FIG. 5 of U.S. Provisional Application No.
61/501,042). The longer transcript initiates SEQ ID NO: 60
(encoding SEQ ID NO: 57). Parts of the genomic sequence transcribed
to produce the longer transcript also act to promote
transcriptional regulatory activity for the shorter transcript.
Various polynucleotide sequences are known in the art which
comprise multiple transcriptional start sites that encode products
targeted to multiple cellular compartments. See for example, Small
(1998) Plant Mol. Biol. 38:265-277 and Thatcher (2007) J of Biol.
Chem. 282:28915-28928. The polypeptide set forth in SEQ ID NO: 57
is localized to the chloroplast, while the polypeptide encoded by
the shorter transcript is localized to the cytosol.
Further provided are variant HPPD proteins as set forth in SEQ ID
NO: 57. "Variant" protein is intended to mean a protein derived
from the native protein by deletion or addition of one or more
amino acids at one or more internal sites in the native protein
and/or substitution of one or more amino acids at one or more sites
in the native protein. Variant proteins encompassed by the present
invention are biologically active, that is they continue to possess
the desired biological activity of the native protein, that is,
HPPD activity and wherein the protein is transported into the
chloroplast of a plant cell. Such variants may result from, for
example, genetic polymorphism or from human manipulation.
Biologically active variants of a HPPD proteins disclosed herein
will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 57 as determined by sequence alignment programs and parameters
described elsewhere herein. A biologically active variant of a
protein of the invention may differ from SEQ ID NO: 57 by as few as
1-15 amino acid residues, as few as 1-10, such as 6-10, as few as
5, as few as 4, 3, 2, or even 1 amino acid residue.
Fragments of amino acid sequences include peptides comprising amino
acid sequences sufficiently identical to or derived from the amino
acid sequence of a HPPD protein, or a partial-length protein and
exhibiting HPPD activity but which include fewer amino acids than
the full-length HPPD-related proteins disclosed herein. A
biologically active portion of a HPPD protein can be a polypeptide
that is, for example, 10, 25, 50, 100, 150, 200 contiguous amino
acids in length, or up to the total number of amino acids present
in a full-length HPPD protein of the current invention (i.e., of
SEQ ID NO: 57). Such biologically active portions can be prepared
by recombinant techniques and evaluated for one or more of the
functional activities of a native HPPD protein, including but not
limited to transport into the chloroplast of a plant cell. As used
herein, a fragment comprises at least 5 contiguous amino acids of
SEQ ID NO: 57. The invention encompasses other fragments, however,
such as any fragment in the protein greater than 6, 7, 8, or 9
amino acids.
The polynucleotide encoding SEQ ID NO: 57 or active fragments and
variants thereof can be provided in an expression cassette for
expression in a plant or organism of interest. The expression
cassette can include 5' and 3' regulatory sequences operably linked
to the polynucleotide of the invention. An operable linkage between
a polynucleotide of interest and a regulatory sequence (i.e., a
promoter) is a functional link that allows for expression of the
polynucleotide of interest. Operably linked elements may be
contiguous or non-contiguous. In some embodiments, the
polynucleotide set forth in SEQ ID NO: 60 can be operably linked to
a heterologous promoter. When used to refer to the joining of two
protein coding regions, by operably linked is intended that the
coding regions are in the same reading frame. The cassette may
additionally contain at least one additional polynucleotide to be
cotransformed into the organism. Alternatively, the additional
polypeptide(s) can be provided on multiple expression cassettes.
Expression cassettes can be provided with a plurality of
restriction sites and/or recombination sites for insertion of the
polynucleotide to be under the transcriptional regulation of the
regulatory regions. The expression cassette may additionally
contain selectable marker genes.
Further provided are plants, plant cells, and seeds having a
heterologous polynucleotide construct comprising an expression
cassette having a promoter operably linked to a polynucleotide
encoding the polypeptide set forth in SEQ ID NO: 57 or an active
variant or fragment thereof, wherein the promoter is heterologous
to said polynucleotide.
VI. Plants
Plants, plant cells, plant parts and seeds, and grain having the
polynucleotide comprising the CTP-encoding sequence operably linked
to a heterologous polynucleotide encoding a polypeptide of interest
are provided. In specific embodiments, the plants and/or plant
parts have stably incorporated at least one of the chimeric
polynucleotides disclosed herein or an active variant or fragment
thereof. Thus, plants, plant cells, plant parts and seed are
provided which comprise at least one polynucleotide comprising a
CTP-encoding sequence operably linked to a heterologous
polynucleotide encoding a polypeptide of interest, wherein the
chloroplast transit peptide comprises any one of SEQ ID NOS: 1, 2,
3, 4, 5, 6, 7, 8, 58 or active variants and fragments thereof, or a
CTP-encoding sequence of any one of the N-terminal regions (about
amino acids 1-53, 1-20, 1-23, 1-17, 1-30, 1-40, 1-60, 1-70, 1-80,
1-85) of an HPPD polypeptide set forth in FIG. 2 or 7 or an active
variant or fragment thereof. Further provided are plants, plant
cells and seeds comprising the HPPD encoding sequences as set forth
in SEQ ID NO: 57 and the polypeptide set forth in SEQ ID NO: 60,
and active variants and fragments thereof.
Further provided are plants, plant cells, plant parts and seeds and
grain having stably incorporated into their genome, the
polynucleotide comprising a CTP-encoding sequence operably linked
to a heterologous polynucleotide encoding a polypeptide of
interest.
In specific embodiments, the chimeric polynucleotide or the HPPD
encoding sequences in the plant or plant part is operably linked to
a constitutive, tissue-preferred, or other promoter for expression
in plants.
As used herein, the term plant includes plant cells, plant
protoplasts, plant cell tissue cultures from which plants can be
regenerated, plant calli, plant clumps, and plant cells that are
intact in plants or parts of plants such as embryos, pollen,
ovules, seeds, leaves, flowers, branches, fruit, kernels, ears,
cobs, husks, stalks, roots, root tips, anthers, and the like. Grain
is intended to mean the mature seed produced by commercial growers
for purposes other than growing or reproducing the species.
Progeny, variants, and mutants of the regenerated plants are also
included within the scope of the invention, provided that these
parts comprise the introduced polynucleotides.
The chimeric polynucleotides, the HPPD encoding sequences and
active variant and fragments thereof disclosed herein may be used
for transformation of any plant species, including, but not limited
to, monocots and dicots. Examples of plant species of interest
include, but are not limited to, corn (Zea mays), Brassica sp.
(e.g., B. napus, B. rapa, B. juncea), particularly those Brassica
species useful as sources of seed oil, alfalfa (Medicago sativa),
rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum
bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum
glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria
italica), finger millet (Eleusine coracana)), sunflower (Helianthus
annuus), safflower (Carthamus tinctorius), wheat (Triticum
aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),
potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton
(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea
batatus), cassava (Manihot esculenta), coffee (Coffea spp.),
coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees
(Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis),
banana (Musa spp.), avocado (Persea americana), fig (Ficus casica),
guava (Psidium guajava), mango (Mangifera indica), olive (Olea
europaea), papaya (Carica papaya), cashew (Anacardium occidentale),
macadamia (Macadamia integrifblia), almond (Prunus amygdalus),
sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats,
barley, vegetables, ornamentals, and conifers.
Vegetables include, but not limited to, tomatoes (Lycopersicon
esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus
vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.),
and members of the genus Cucumis such as cucumber (C. sativus),
cantaloupe (C. cantalupensis), and musk melon (C. melo).
Ornamentals include, but not limited to, azalea (Rhododendron
spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus
rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils
(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus
caryophyllus), poinsettia (Euphorbia pukherrima), and
chrysanthemum.
Conifers that may be employed in practicing the present invention
include, for example, pines such as loblolly pine (Pinus taeda),
slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),
lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata);
Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga
canadensis); Sitka spruce (Picea glauca); redwood (Sequoia
sempervirens); true firs such as silver fir (Abies amabilis) and
balsam fir (Abies balsamea); and cedars such as Western red cedar
(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis
nootkatensis), and Poplar and Eucalyptus. In specific embodiments,
plants of the present invention are crop plants (for example, corn,
alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,
sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn
and soybean plants are optimal, and in yet other embodiments corn
plants are optimal.
Other plants of interest include grain plants that provide seeds of
interest, oil-seed plants, and leguminous plants. Seeds of interest
include grain seeds, such as corn, wheat, barley, rice, sorghum,
rye, etc. Oil-seed plants include cotton, soybean, safflower,
sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous
plants include beans and peas. Beans include guar, locust bean,
fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava
bean, lentils, chickpea, etc.
In some embodiments, the polynucleotides comprising the
CTP-encoding sequence operably linked to the polynucleotide
encoding the polypeptide of interest are engineered into a
molecular stack. Thus, the various plants, plant cells and seeds
disclosed herein can further comprise one or more traits of
interest, and in more specific embodiments, the plant, plant part
or plant cell is stacked with any combination of polynucleotide
sequences of interest in order to create plants with a desired
combination of traits. As used herein, the term "stacked" includes
having the multiple traits present in the same plant.
These stacked combinations can be created by any method including,
but not limited to, breeding plants by any conventional
methodology, or genetic transformation. If the sequences are
stacked by genetically transforming the plants, the polynucleotide
sequences of interest can be combined at any time and in any order.
The traits can be introduced simultaneously in a co-transformation
protocol with the polynucleotides of interest provided by any
combination of transformation cassettes. For example, if two
sequences will be introduced, the two sequences can be contained in
separate transformation cassettes (trans) or contained on the same
transformation cassette (cis). Expression of the sequences can be
driven by the same promoter or by different promoters. In certain
cases, it may be desirable to introduce a transformation cassette
that will suppress the expression of the polynucleotide of
interest. This may be combined with any combination of other
suppression cassettes or overexpression cassettes to generate the
desired combination of traits in the plant. It is further
recognized that polynucleotide sequences can be stacked at a
desired genomic location using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference.
A "subject plant or plant cell" is one in which genetic alteration,
such as transformation, has been affected as to a gene of interest,
or is a plant or plant cell which is descended from a plant or cell
so altered and which comprises the alteration. A "control" or
"control plant" or "control plant cell" provides a reference point
for measuring changes in phenotype of the subject plant or plant
cell.
A control plant or plant cell may comprise, for example: (a) a
wild-type plant or cell, i.e., of the same genotype as the starting
material for the genetic alteration which resulted in the subject
plant or cell; (b) a plant or plant cell of the same genotype as
the starting material but which has been transformed with a null
construct (i.e. with a construct which has no known effect on the
trait of interest, such as a construct comprising a marker gene);
(c) a plant or plant cell which is a non-transformed segregant
among progeny of a subject plant or plant cell; (d) a plant or
plant cell genetically identical to the subject plant or plant cell
but which is not exposed to conditions or stimuli that would induce
expression of the gene of interest; or (e) the subject plant or
plant cell itself, under conditions in which the gene of interest
is not expressed.
VII. Polynucleotide Constructs
The use of the term "polynucleotide" is not intended to limit the
present invention to polynucleotides comprising DNA. Those of
ordinary skill in the art will recognize that polynucleotides can
comprise ribonucleotides and combinations of ribonucleotides and
deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides
include both naturally occurring molecules and synthetic analogues.
The polynucleotides of the invention also encompass all forms of
sequences including, but not limited to, single-stranded forms,
double-stranded forms, hairpins, stem-and-loop structures, and the
like.
The chimeric polynucleotides or the HPPD encoding sequences
disclosed herein can be provided in expression cassettes for
expression in the plant of interest. The cassette can include 5'
and 3' regulatory sequences operably linked to the chimeric
polynucleotide or active variant or fragment thereof. "Operably
linked" is intended to mean a functional linkage between two or
more elements. For example, an operably linkage between a
polynucleotide of interest and a regulatory sequence (i.e., a
promoter) is a functional link that allows for expression of the
polynucleotide of interest. Operably linked elements may be
contiguous or non-contiguous. When used to refer to the joining of
two protein coding regions, by operably linked is intended that the
coding regions are in the same reading frame. The cassette may
additionally contain at least one additional gene to be
cotransformed into the organism. Alternatively, the additional
gene(s) can be provided on multiple expression cassettes. Such an
expression cassette is provided with a plurality of restriction
sites and/or recombination sites for insertion of the chimeric
polynucleotide or active variant or fragment thereof to be under
the transcriptional regulation of the regulatory regions. The
expression cassette may additionally contain selectable marker
genes.
The expression cassette can include in the 5'-3' direction of
transcription, a transcriptional and translational initiation
region (i.e., a promoter), a CTP-encoding sequence or active
variant or fragment thereof operably linked to a polynucleotide
encoding a polypeptide of interest and a transcriptional and
translational termination region (i.e., termination region)
functional in plants. The regulatory regions (i.e., promoters,
transcriptional regulatory regions, and translational termination
regions) and/or the CTP-encoding sequence and/or the polynucleotide
encoding the polypeptide of interest may be native/analogous to the
host cell or to each other. Alternatively, the regulatory regions
and/or the CTP-encoding sequence and/or the polynucleotide encoding
the polypeptide of interest may be heterologous to the host cell or
to each other. In specific embodiments, the CTP-encoding sequenced
is operably linked to the 5' end of the polynucleotide of interest,
such that, in the resulting chimeric polypeptide, the CTP is
operably linked to the N-terminal region of the polypeptide of
interest.
As used herein, "heterologous" in reference to a sequence is a
sequence that originates from a foreign species, or, if from the
same species, is substantially modified from its native form in
composition and/or genomic locus by deliberate human intervention.
For example, a promoter operably linked to a heterologous
polynucleotide is from a species different from the species from
which the polynucleotide was derived, or, if from the
same/analogous species, one or both are substantially modified from
their original form and/or genomic locus, or the promoter is not
the native promoter for the operably linked polynucleotide.
The termination region may be native with the transcriptional
initiation region, may be native with the operably linked
polynucleotide sequence of interest, may be native with the plant
host, or may be derived from another source (i.e., foreign or
heterologous) to the promoter, the CTP, the polynucleotide sequence
of interest, the plant host, or any combination thereof. Convenient
termination regions are available from the Ti-plasmid of A.
tumefaciens, such as the octopine synthase and nopaline synthase
termination regions. See also Guerineau et al. (1991) Mol. Gen.
Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et
al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell
2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al.
(1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987)
Nucleic Acids Res. 15:9627-9639.
Where appropriate, the polynucleotides may be optimized for
increased expression in the transformed plant. That is, the
polynucleotides can be synthesized using plant-preferred codons for
improved expression. See, for example, Campbell and Gowri (1990)
Plant Physiol. 92:1-11 for a discussion of host-preferred codon
usage. Methods are available in the art for synthesizing
plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831,
and 5,436,391, and Murray et al. (1989) Nucleic Acids Res.
17:477-498, herein incorporated by reference.
Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other such
well-characterized sequences that may be deleterious to gene
expression. The G-C content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. When possible,
the sequence is modified to avoid predicted hairpin secondary mRNA
structures.
The expression cassettes may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci.
USA 86:6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238),
MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and
human immunoglobulin heavy-chain binding protein (BiP) (Macejak et
al. (1991) Nature 353:90-94); untranslated leader from the coat
protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al.
(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV)
(Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,
New York), pp. 237-256); and maize chlorotic mottle virus leader
(MCMV) (Lommel et al. (1991) Virology 81:382-385. See also,
Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.
In preparing the expression cassette, the various DNA fragments may
be manipulated, so as to provide for the DNA sequences in the
proper orientation and, as appropriate, in the proper reading
frame. Toward this end, adapters or linkers may be employed to join
the DNA fragments or other manipulations may be involved to provide
for convenient restriction sites, removal of superfluous DNA,
removal of restriction sites, or the like. For this purpose, in
vitro mutagenesis, primer repair, restriction, annealing,
resubstitutions, e.g., transitions and transversions, may be
involved.
A number of promoters can be used to express the various sequences
of interest including the native promoter of the polynucleotide
sequence of interest. The promoters can be selected based on the
desired outcome. Such promoters include, for example, constitutive,
tissue-preferred, or other promoters for expression in plants.
Constitutive promoters include, for example, the core promoter of
the Rsyn7/synthetic core 11 promoter and other constitutive
promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the
core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812);
rice actin (McElroy et al. (1990) Plant Ce112:163-171); ubiquitin
(Christensen et al. (1989) Plant Mol. Bioi. 12:619-632 and
Christensen et al. 20 (1992) Plant Mol. Bioi. 18:675-689); pEMU
(Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et
al. (1984) EMBO J 3:2723-2730); ALS promoter (U.S. Pat. No.
5,659,026), and the like. Other constitutive promoters include, for
example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
Additional promoters of interest are set forth in U.S. Utility
application Ser. No. 13/209,017, now issued U.S. Pat. No.
8,993,837, entitled "Chimeric Promoters And Methods of Use" filed
concurrently herewith and herein incorporated by reference in its
entirety.
Tissue-preferred promoters can be utilized to target enhanced HPPD
expression within a particular plant tissue. Tissue-preferred
promoters include those described in Yamamoto et al. (1997) Plant
J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol.
38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343;
Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al.
(1996) Plant Physiol. 112(3):1331-1341; Van Camp et al. (1996)
Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant
Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters
can be modified, if necessary, for weak expression.
Leaf-preferred promoters are known in the art. See, for example,
Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994)
Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
Synthetic promoters can be used to express the polynucleotide
sequences of interest or biologically active variants and fragments
thereof.
The expression cassette can also comprise a selectable marker gene
for the selection of transformed cells. Selectable marker genes are
utilized for the selection of transformed cells or tissues. Marker
genes include genes encoding antibiotic resistance, such as those
encoding neomycin phosphotransferase II (NEO) and hygromycin
phosphotransferase (HPT), as well as genes conferring resistance to
herbicidal compounds, such as glyphosate, glufosinate ammonium,
bromoxynil, sulfonylureas, dicamba, and 2,4-dichlorophenoxyacetate
(2,4-D). Additional selectable markers include phenotypic markers
such as .beta.-galactosidase and fluorescent proteins such as green
fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng
85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan
florescent protein (CYP) (Bolte et al. (2004) J Cell Science
117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and
yellow florescent protein (PhiYFP.TM. from Evrogen, see, Bolte et
al. (2004) J. Cell Science 117:943-54). For additional selectable
markers, see generally, Yarranton (1992) Curr. Opin. Biotech.
3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA
89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992)
Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,
pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987)
Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et
al. (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al.
(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al.
(1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University
of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA
90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356;
Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956;
Bairn et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076;
Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595;
Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993)
Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc.
Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob.
Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of
Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill
et al. (1988) Nature 334:721-724. Such disclosures are herein
incorporated by reference. The above list of selectable marker
genes is not meant to be limiting. Any selectable marker gene can
be used in the present invention, including for example, DsRed as
described in Example 3, 4, 6 or 9 and FIG. 5.
IIX. Method of Introducing
Various methods can be used to introduce a sequence of interest
into a plant or plant part. "Introducing" is intended to mean
presenting to the plant, plant cell or plant part the
polynucleotide or polypeptide in such a manner that the sequence
gains access to the interior of a cell of the plant. The methods of
the invention do not depend on a particular method for introducing
a sequence into a plant or plant part, only that the polynucleotide
or polypeptides gains access to the interior of at least one cell
of the plant. Methods for introducing polynucleotide or
polypeptides into plants are known in the art including, but not
limited to, stable transformation methods, transient transformation
methods, and virus-mediated methods.
"Stable transformation" is intended to mean that the nucleotide
construct introduced into a plant integrates into the genome of the
plant and is capable of being inherited by the progeny thereof
"Transient transformation" is intended to mean that a
polynucleotide is introduced into the plant and does not integrate
into the genome of the plant or a polypeptide is introduced into a
plant.
Transformation protocols as well as protocols for introducing
polypeptides or polynucleotide sequences into plants may vary
depending on the type of plant or plant cell, i.e., monocot or
dicot, targeted for transformation. Suitable methods of introducing
polypeptides and polynucleotides into plant cells include
microinjection (Crossway et al. (1986) Biotechniques 4:320-334),
electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA
83:5602-5606, Agrobacterium-mediated transformation (U.S. Pat. No.
5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic
particle acceleration (see, for example, U.S. Pat. No. 4,945,050;
U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and, 5,932,782;
Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:
Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,
Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lecl
transformation (WO 00/28058). Also see Weissinger et al. (1988)
Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate
Science and Technology 5:27-37 (onion); Christou et al. (1988)
Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)
Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In
Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998)
Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990)
Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl.
Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)
Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855;
5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol.
91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839
(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)
311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al.
(1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet
et al. (1985) in The Experimental Manipulation of Ovule Tissues,
ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler
et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al.
(1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated
transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505
(electroporation); Li et al. (1993) Plant Cell Reports 12:250-255
and Christou and Ford (1995) Annals of Botany 75:407-413 (rice);
Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via
Agrobacterium tumefaciens); all of which are herein incorporated by
reference.
In specific embodiments, the polynucleotide comprising the
CTP-encoding sequence operably linked to a heterologous
polynucleotide encoding the polypeptide of interest or the sequence
encoding the HPPD polypeptide can be provided to a plant using a
variety of transient transformation methods. Such transient
transformation methods include, but are not limited to, the
introduction of the protein or active variants and fragments
thereof directly into the plant. Such methods include, for example,
microinjection or particle bombardment. See, for example, Crossway
et al. (1986) Mol. Gen. Genet. 202:179-185; Nomura et al. (1986)
Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci.
91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science
107:775-784, all of which are herein incorporated by reference.
In other embodiments, the polynucleotide may be introduced into
plants by contacting plants with a virus or viral nucleic acids.
Generally, such methods involve incorporating a nucleotide
construct of the invention within a DNA or RNA molecule. It is
recognized that a protein sequence may be initially synthesized as
part of a viral polyprotein, which later may be processed by
proteolysis in vivo or in vitro to produce the desired recombinant
protein. Further, it is recognized that promoters of the invention
also encompass promoters utilized for transcription by viral RNA
polymerases. Methods for introducing polynucleotides into plants
and expressing a protein encoded therein, involving viral DNA or
RNA molecules, are known in the art. See, for example, U.S. Pat.
Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and
Porta et al. (1996) Molecular Biotechnology 5:209-221; herein
incorporated by reference.
Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference. Briefly, the polynucleotide of the invention can be
contained in a transfer cassette flanked by two non-recombinogenic
recombination sites. The transfer cassette is introduced into a
plant having stably incorporated into its genome a target site
which is flanked by two non-recombinogenic recombination sites that
correspond to the sites of the transfer cassette. An appropriate
recombinase is provided and the transfer cassette is integrated at
the target site. The polynucleotide of interest is thereby
integrated at a specific chromosomal position in the plant genome.
Other methods to target polynucleotides are set forth in WO
2009/114321 (herein incorporated by reference), which describes
"custom" meganucleases produced to modify plant genomes, in
particular the genome of maize. See, also, Gao et al. (2010) Plant
Journal 1:176-187.
The cells that have been transformed may be grown into plants in
accordance with conventional ways. See, for example, McCormick et
al. (1986) Plant Cell Reports 5:81-84. These plants may then be
grown, and either pollinated with the same transformed strain or
different strains, and the resulting progeny having constitutive
expression of the desired phenotypic characteristic identified. Two
or more generations may be grown to ensure that expression of the
desired phenotypic characteristic is stably maintained and
inherited and then seeds harvested to ensure expression of the
desired phenotypic characteristic has been achieved. In this
manner, the present invention provides transformed seed (also
referred to as "transgenic seed") having a polynucleotide of the
invention, for example, an expression cassette of the invention,
stably incorporated into their genome.
IX. Methods of Use
Methods of the present invention are directed to the proper
expression, translocation, and processing of chloroplast-targeted
sequences in plants and plant cells under the control of the CTP
sequences disclosed herein. For the purposes of the present
invention, a "processed" chloroplast targeted protein is one in
which the CTP has been removed. At the time of translocation of a
chloroplast targeted protein into the chloroplast of a plant cell,
the CTP is removed from the targeted protein by cleavage at a
particular "cleavage site" between the CTP and the mature protein.
The cleavage site can be determined experimentally, or may be
predicted based on sequence structure (e.g., by alignment of the
unprocessed protein with chloroplast targeted proteins in which the
cleavage site is known, by analyzing the sequence for the presence
of characteristic CTP domains, and the like) or by using one or
more algorithms for cleavage site prediction as discussed elsewhere
herein (e.g., SignalP).
Thus, methods for targeting a polypeptide of interest to the
chloroplast are provided. Such methods comprise introducing a
chimeric polynucleotide comprising a CTP-encoding sequence operably
linked to a polynucleotide encoding a polypeptide of interest into
a plant cell and expressing the chimeric polynucleotide in the
plant cell.
Depending on the polypeptide of interest targeted to the
chloroplast, the transgenic plants may have a change in phenotype,
including, but not limited to, an altered pathogen or insect
defense mechanism, an increased resistance to one or more
herbicides, an increased ability to withstand stressful
environmental conditions, a modified ability to produce starch, a
modified level of starch production, a modified oil content and/or
composition, a modified ability to utilize, partition and/or store
nitrogen, and the like. These results can be achieved through the
expression and targeting of a polypeptide of interest to
chloroplasts in plants, wherein the polypeptide of interest
functions in the chloroplast. The CTP sequences of the invention
are useful for targeting native sequences as well as heterologous
(non-native) sequences in plants.
X. Stacking Other Traits of Interest
In specific embodiments, the HPPD polynucleotides or active
variants and fragments thereof disclosed herein or the various
sequences encoding the chimeric polypeptides are engineered into a
molecular stack. Thus, the various plants, plant cells and seeds
disclosed herein can further comprise one or more traits of
interest, and in more specific embodiments, the plant, plant part
or plant cell is stacked with any combination of polynucleotide
sequences of interest in order to create plants with a desired
combination of traits. As used herein, the term "stacked" includes
having the multiple traits present in the same plant (i.e., both
traits are incorporated into the nuclear genome, one trait is
incorporated into the nuclear genome and one trait is incorporated
into the genome of a plastid, or both traits are incorporated into
the genome of a plastid). In one non-limiting example, "stacked
traits" comprise a molecular stack where the sequences are
physically adjacent to each other. A trait, as used herein, refers
to the phenotype derived from a particular sequence or groups of
sequences. In one embodiment, the molecular stack comprises at
least one additional polynucleotide that also confers tolerance to
at least one HPPD inhibitor and/or at least one additional
polynucleotide that confers tolerance to a second herbicide.
Thus, in one embodiment, the plants, plant cells or plant part
having the HPPD polynucleotide or active variants or fragments
thereof disclosed herein or a sequence encoding the chimeric
polypeptides is stacked with at least one other HPPD sequence. Such
HPPD sequence include the HPPD sequence and variants and fragment
thereof disclosed herein, as well as other HPPD sequence, which
include but are not limited to the HPPD sequences set forth in U.S.
Pat. Nos. 6,245,968 B1; 6,268,549; and 6,069,115; international
publication WO 99/23886, each of which is herein incorporated by
reference, and those disclosed in U.S. Utility application Ser. No.
13/208,966 entitled "Compositions and Methods Comprising Sequences
Having Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity" filed
concurrently herewith and incorporated by reference in its
entirety.
In still other embodiments, plants, plant cells, explants and
expression cassettes comprising the HPPD sequences, the various
sequences encoding the chimeric polypeptides, or active variants
and fragments thereof are stacked with a sequence that confers
tolerance to HPPD inhibitors through a different mechanism than the
HPPD polypeptide. For example, a P450 sequence could be employed
which provides tolerance to HPPD-inhibitors by metabolism of the
herbicide. Such sequences including, but are not limited to, the
NSF1 gene. See, US 2007/0214515 and US 2008/0052797 both of which
are herein incorporated by reference in their entirety.
Known genes that confer tolerance to herbicides such as e.g.,
auxin, HPPD, glyphosate, dicamba, glufosinate, sulfonylurea,
bromoxynil and norflurazon herbicides can be stacked either as a
molecular stack or a breeding stack with plants expressing the
traits disclosed herein. Polynucleotide molecules encoding proteins
involved in herbicide tolerance include, but are not limited to, a
polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS) disclosed in U.S. Pat. Nos. 39,247; 6,566,587 for
imparting glyphosate tolerance; polynucleotide molecules encoding a
glyphosate oxidoreductase (GOX) disclosed in U.S. Pat. No.
5,463,175 and a glyphosate-N-acetyl transferase (GAT) disclosed in
U.S. Pat. Nos. 7,622,641; 7,462,481; 7,531,339; 7,527,955;
7,709,709; 7,714,188 and 7,666,643 also for providing glyphosate
tolerance; dicamba monooxygenase disclosed in U.S. Pat. No.
7,022,896 and WO2007146706A2 for providing dicamba tolerance; a
polynucleotide molecule encoding AAD12 disclosed in U.S. Pat. App.
Pub. No. 2005731044 or WO2007053482A2 or encoding AAD1 disclosed in
US20110124503A1 or U.S. Pat. No. 7,838,733 for providing tolerance
to auxin herbicides (2,4-D); a polynucleotide molecule encoding
hydroxyphenylpyruvate dioxygenase (HPPD) for providing tolerance to
HPPD inhibitors (e.g., hydroxyphenylpyruvate dioxygenase) disclosed
in e.g., U.S. Pat. No. 7,935,869; US20090055976A1; and
US20110023180A1; each publication is herein incorporated by
reference in its entirety.
In some embodiments, the plant or plant cells having the HPPD
polynucleotides, the various sequences encoding the chimeric
polypeptides or active variants or fragments thereof may be stacked
with other herbicide-tolerance traits to create a transgenic plant
of the invention with further improved properties. Other
herbicide-tolerance polynucleotides that could be used in such
embodiments include those conferring tolerance to glyphosate such
as, for example, glyphosate N-acetyltransferase. See, for example,
WO02/36782, US Publication 2004/0082770 and WO 2005/012515, U.S.
Pat. No. 7,462,481, U.S. Pat. No. 7,405,074, each of which is
herein incorporated by reference.
Additional glyphosate-tolerance traits include a sequence that
encodes a glyphosate oxido-reductase enzyme as described more fully
in U.S. Pat. Nos. 5,776,760 and 5,463,175. Other traits that could
be combined with the HPPD sequence disclosed herein include those
derived from polynucleotides that confer on the plant the capacity
to produce a higher level or glyphosate insensitive
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), for example,
as more fully described in U.S. Pat. Nos. 6,248,876 B1; 5,627,061;
5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;
4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;
4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287
E; and 5,491,288; and international publications WO 97/04103; WO
00/66746; WO 01/66704; and WO 00/66747. Other traits that could be
combined with the HPPD sequences disclosed herein include those
conferring tolerance to sulfonylurea and/or imidazolinone, for
example, as described more fully in U.S. Pat. Nos. 5,605,011;
5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373;
5,331,107; 5,928,937; and 5,378,824; and international publication
WO 96/33270.
In other embodiments, the plants or plant cell or plant part having
the HPPD sequence or an active variant or fragment thereof is
stacked with, for example, a sequence which confers tolerance to an
ALS inhibitor. As used herein, an "ALS inhibitor-tolerant
polypeptide" comprises any polypeptide which when expressed in a
plant confers tolerance to at least one ALS inhibitor. A variety of
ALS inhibitors are known and include, for example, sulfonylurea,
imidazolinone, triazolopyrimidines, pryimidinyoxy(thio)benzoates,
and/or sulfonylaminocarbonyltriazolinone herbicides. Additional ALS
inhibitors are known and are disclosed elsewhere herein. It is
known in the art that ALS mutations fall into different classes
with regard to tolerance to sulfonylureas, imidazolinones,
triazolopyrimidines, and pyrimidinyl(thio)benzoates, including
mutations having the following characteristics: (1) broad tolerance
to all four of these groups; (2) tolerance to imidazolinones and
pyrimidinyl(thio)benzoates; (3) tolerance to sulfonylureas and
triazolopyrimidines; and (4) tolerance to sulfonylureas and
imidazolinones.
Various ALS inhibitor-tolerant polypeptides can be employed. In
some embodiments, the ALS inhibitor-tolerant polynucleotides
contain at least one nucleotide mutation resulting in one amino
acid change in the ALS polypeptide. In specific embodiments, the
change occurs in one of seven substantially conserved regions of
acetolactate synthase. See, for example, Hattori et al. (1995)
Molecular Genetics and Genomes 246:419-425; Lee et al. (1998) EMBO
Journal 7:1241-1248; Mazur et al. (1989) Ann. Rev. Plant Phys.
40:441-470; and U.S. Pat. No. 5,605,011, each of which is
incorporated by reference in their entirety. The ALS
inhibitor-tolerant polypeptide can be encoded by, for example, the
SuRA or SuRB locus of ALS. In specific embodiments, the ALS
inhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA
ALS mutant, the S4 mutant or the S4/HRA mutant or any combination
thereof. Different mutations in ALS are known to confer tolerance
to different herbicides and groups (and/or subgroups) of
herbicides; see, e.g., Tranel and Wright (2002) Weed Science
50:700-712. See also, U.S. Pat. Nos. 5,605,011, 5,378,824,
5,141,870, and 5,013,659, each of which is herein incorporated by
reference in their entirety. The soybean, maize, and Arabidopsis
HRA sequences are disclosed, for example, in WO2007/024782, herein
incorporated by reference.
In some embodiments, the ALS inhibitor-tolerant polypeptide confers
tolerance to sulfonylurea and imidazolinone herbicides. The
production of sulfonylurea-tolerant plants and
imidazolinone-tolerant plants is described more fully in U.S. Pat.
Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180;
5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and
international publication WO 96/33270, which are incorporated
herein by reference in their entireties for all purposes. In
specific embodiments, the ALS inhibitor-tolerant polypeptide
comprises a sulfonamide-tolerant acetolactate synthase (otherwise
known as a sulfonamide-tolerant acetohydroxy acid synthase) or an
imidazolinone-tolerant acetolactate synthase (otherwise known as an
imidazolinone-tolerant acetohydroxy acid synthase).
In further embodiments, the plants or plant cell or plant part
having the HPPD sequence or an active variant or fragment thereof
is stacked with, for example, a sequence which confers tolerance to
an ALS inhibitor and glyphosate tolerance. In one embodiment, the
HPPD sequence or active variant or fragment thereof is stacked with
HRA and a glyphosate N-acetyltransferase. See, WO2007/024782,
2008/0051288 and WO 2008/112019, each of which is herein
incorporated by reference.
In still other embodiments, the plant or plant cell or plant part
having the HPPD sequence or an active variant or fragment thereof
may be stacked with, for example, aryloxyalkanoate dioxygenase
polynucleotides (which confer tolerance to 2,4-D and other phenoxy
auxin herbicides as well as to aryloxyphenoxypropionate herbicides
as described, for example, in WO2005/107437) and dicamba-tolerance
polynucleotides as described, for example, in Herman et al. (2005)
J. Biol. Chem. 280: 24759-24767, auxin polypeptides and an acetyl
coenzyme A carboxylase (ACCase) polypeptides.
Other examples of herbicide-tolerance traits that could be combined
with the plant or plant cell or plant part having the HPPD
sequence, the various sequences encoding the chimeric polypeptides
or an active variants or fragments thereof include those conferred
by polynucleotides encoding an exogenous phosphinothricin
acetyltransferase, as described in U.S. Pat. Nos. 5,969,213;
5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;
5,646,024; 6,177,616; and 5,879,903. Plants containing an exogenous
phosphinothricin acetyltransferase can exhibit improved tolerance
to glufosinate herbicides, which inhibit the enzyme glutamine
synthase. Other examples of herbicide-tolerance traits that could
be combined with the plants or plant cell or plant part having the
HPPD sequence or an active variant or fragment thereof include
those conferred by polynucleotides conferring altered
protoporphyrinogen oxidase (protox) activity, as described in U.S.
Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373; and
international publication WO 01/12825. Plants containing such
polynucleotides can exhibit improved tolerance to any of a variety
of herbicides which target the protox enzyme (also referred to as
"protox inhibitors").
Other examples of herbicide-tolerance traits that could be combined
with the plants or plant cell or plant part having the HPPD
sequence, the various sequences encoding the chimeric polypeptides,
or an active variant or fragment thereof include those conferring
tolerance to at least one herbicide in a plant such as, for
example, a maize plant or horseweed. Herbicide-tolerant weeds are
known in the art, as are plants that vary in their tolerance to
particular herbicides. See, e.g., Green and Williams (2004)
"Correlation of Corn (Zea mays) Inbred Response to Nicosulfuron and
Mesotrione," poster presented at the WSSA Annual Meeting in Kansas
City, Mo., Feb. 9-12, 2004; Green (1998) Weed Technology 12:
474-477; Green and Ulrich (1993) Weed Science 41: 508-516. The
trait(s) responsible for these tolerances can be combined by
breeding or via other methods with the plants or plant cell or
plant part having the HPPD sequence, the various sequences encoding
the chimeric polypeptides or an active variants or fragments
thereof to provide a plant of the invention as well as methods of
use thereof.
In still further embodiments, the HPPD sequences, the various
sequences encoding the chimeric polypeptides or active variants or
fragments thereof can be stacked with at least one polynucleotide
encoding a homogentisate solanesyltransferase (HST). See, for
example, WO2010023911 herein incorporated by reference in its
entirety. In such embodiments, classes of herbicidal
compounds--which act wholly or in part by inhibiting HST can be
applied over the plants having the HTS polypeptide.
The plant or plant cell or plant part having the HPPD sequence, the
various sequences encoding the chimeric polypeptides, or an active
variants or fragments thereof can also be combined with at least
one other trait to produce plants that further comprise a variety
of desired trait combinations including, but not limited to, traits
desirable for animal feed such as high oil content (e.g., U.S. Pat.
No. 6,232,529); balanced amino acid content (e.g., hordothionins
(U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409;
U.S. Pat. No. 5,850,016); barley high lysine (Williamson et al.
(1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122) and high
methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:
6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al.
(1989) Plant Mol. Biol. 12:123)); increased digestibility (e.g.,
modified storage proteins (U.S. application Ser. No. 10/053,410,
filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No.
10/005,429, filed Dec. 3, 2001)); the disclosures of which are
herein incorporated by reference. Desired trait combinations also
include LLNC (low linolenic acid content; see, e.g., Dyer et al.
(2002) Appl. Microbiol. Biotechnol. 59: 224-230) and OLCH (high
oleic acid content; see, e.g., Fernandez-Moya et al. (2005) J.
Agric. Food Chem. 53: 5326-5330).
The plant or plant cell or plant part having the HPPD sequence, the
various sequences encoding the chimeric polypeptides or an active
variants or fragments thereof can also be combined with other
desirable traits such as, for example, fumonisim detoxification
genes (U.S. Pat. No. 5,792,931), avirulence and disease resistance
genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993)
Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089), and
traits desirable for processing or process products such as
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE), and starch debranching enzymes (SDBE));
and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.
170:5837-5847) facilitate expression of polyhydroxyalkanoates
(PHAs)); the disclosures of which are herein incorporated by
reference. One could also combine herbicide-tolerant
polynucleotides with polynucleotides providing agronomic traits
such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk
strength, flowering time, or transformation technology traits such
as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO
00/17364, and WO 99/25821); the disclosures of which are herein
incorporated by reference.
In other embodiments, the plant or plant cell or plant part having
the HPPD sequence, the various sequences encoding the chimeric
polypeptides, or an active variants or fragments thereof may be
stacked with any other polynucleotides encoding polypeptides having
pesticidal and/or insecticidal activity, such as Bacillus
thuringiensis toxic proteins (described in U.S. Pat. Nos.
5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et
al. (1986) Gene 48: 109; Lee et al. (2003) Appl. Environ.
Microbiol. 69: 4648-4657 (Vip3A); Galitzky et al. (2001) Acta
Crystallogr. D. Biol. Crysiallogr. 57: 1101-1109 (Cry3Bbl); and
Herman et al. (2004) J. Agric. Food Chem. 52: 2726-2734 (CrylF)),
lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825, pentin
(described in U.S. Pat. No. 5,981,722), and the like. The
combinations generated can also include multiple copies of any one
of the polynucleotides of interest.
In another embodiment, the plant or plant cell or plant part having
the HPPD sequence, the various sequences encoding the chimeric
polypeptides, or an active variant or fragment thereof can also be
combined with the Rcg1 sequence or biologically active variant or
fragment thereof. The Rcg1 sequence is an anthracnose stalk rot
resistance gene in corn. See, for example, U.S. patent application
Ser. No. 11/397,153, Ser. No. 11/397,275, and Ser. No. 11/397,247,
each of which is herein incorporated by reference.
These stacked combinations can be created by any method including,
but not limited to, breeding plants by any conventional
methodology, or genetic transformation. If the sequences are
stacked by genetically transforming the plants, the polynucleotide
sequences of interest can be combined at any time and in any order.
The traits can be introduced simultaneously in a co-transformation
protocol with the polynucleotides of interest provided by any
combination of transformation cassettes. For example, if two
sequences will be introduced, the two sequences can be contained in
separate transformation cassettes (trans) or contained on the same
transformation cassette (cis). Expression of the sequences can be
driven by the same promoter or by different promoters. In certain
cases, it may be desirable to introduce a transformation cassette
that will suppress the expression of the polynucleotide of
interest. This may be combined with any combination of other
suppression cassettes or overexpression cassettes to generate the
desired combination of traits in the plant. It is further
recognized that polynucleotide sequences can be stacked at a
desired genomic location using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference.
Non-limiting embodiment include:
1. A chimeric polynucleotide comprising a nucleotide sequence
encoding a chloroplast transit peptide operably linked to a
heterologous polynucleotide encoding a polypeptide of interest,
wherein said chloroplast transit peptide comprises
a) an amino acid sequence comprising the amino acids of SEQ ID
NO:1;
b) an amino acid sequence having at least 90% sequence identity to
SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 58;
c) an amino acid sequence having at least 17 consecutive amino
acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, or 58; or,
d) an amino acid sequence having at least 90% sequence identity to
SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 58 and having at least 17
consecutive amino acids of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, or
58.
2. The chimeric polynucleotide of embodiment 1, wherein said
chloroplast transit peptide comprises SEQ ID NO: 2, 3, 4, 5, 6, 7,
8, or 58.
3. The chimeric polynucleotide of embodiment 1 or 2, wherein said
polypeptide of interest comprises a 4-hydroxphenylpyruvate
dioxygenase (HPPD) polypeptide having HPPD activity.
4. A nucleic acid construct comprising the chimeric polynucleotide
of any one of embodiments 1-3.
5. The nucleic acid construct of embodiment 4, further comprising a
promoter operably linked to said chimeric polynucleotide.
6. A cell comprising at least one chimeric polynucleotide of any of
embodiments 1-3 or the nucleic acid construct of any one of
embodiments 4 or 5.
7. The cell of embodiment 6, wherein said cell is a plant cell.
8. The cell of embodiment 7, wherein said polynucleotide or nucleic
acid construct is stably incorporated into the genome of said plant
cell.
9. The cell of any one of embodiments 7-8, wherein said plant cell
is from a monocot.
10. The cell of embodiment 9, wherein said monocot is maize, wheat,
rice, barley, sorghum, or rye.
11. The cell of any one of embodiments 7-8, wherein said plant cell
is from a dicot.
12. The cell of embodiment 11, wherein the dicot is soybean,
Brassica, sunflower, cotton, or alfalfa.
13. A plant comprising at least one plant cell of any one of
embodiments 7-12.
14. A plant explant comprising at least one plant cell of any one
of embodiments 7-12.
15. A transgenic seed produced by the plant of embodiment 13.
16. The plant, plant cell, or seed of any one of 11-15, wherein the
plant, plant cell, or seed further comprises at least one
polypeptide imparting tolerance to a herbicide.
17. The plant, plant cell, or seed of embodiment 16, wherein said
at least one polypeptide imparting tolerance to a herbicide
comprises: (a) a sulfonylurea-tolerant acetolactate synthase; (b)
an imidazolinone-tolerant acetolactate synthase; (c) a
glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase;
(d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a
glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl
transferase; (g) a protoporphyrinogen oxidase. (h) an auxin enzyme
or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A
carboxylase (ACCase).
18. A chimeric polypeptide encoded by the polynucleotide of any one
of embodiments 1-3.
19. A method of targeting a polypeptide of interest to a
chloroplast comprising expressing a chimeric polynucleotide of any
one of embodiments 1-3 or the nucleic acid construct of embodiment
4 or 5 in a plant cell.
20. A method of targeting a polypeptide of interest to a
chloroplast comprising introducing the chimeric polynucleotide of
any one of embodiments 1-3 or the nucleic acid construct of
embodiment 4 or 5 in a plant cell and expressing said chimeric
polynucleotide in the plant cell.
21. The method of embodiment 19 or 20, wherein said method further
comprises regenerating a transgenic plant from said plant cell.
22. The method of any one of embodiments 19-21, wherein said plant
cell is from a dicot.
23. The method of embodiment 22, wherein said dicot is selected
from the group consisting of soybean, Brassica, sunflower, cotton,
or alfalfa.
24. The method of any one of embodiments 19-21, wherein said plant
cell is from a monocot.
25. The method of embodiment 24, wherein said dicot is selected
from the group consisting of maize, wheat, rice, barley, sorghum,
or rye.
26. The method of any one of embodiments 19-25, wherein the plant
cell further comprises at least one polypeptide imparting tolerance
to a herbicide.
27. The plant, plant cell, or seed of embodiment 26, wherein said
at least one polypeptide imparting tolerance to a herbicide
comprises: (a) a sulfonylurea-tolerant acetolactate synthase; (b)
an imidazolinone-tolerant acetolactate synthase; (c) a
glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase;
(d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a
glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl
transferase; (g) a protoporphyrinogen oxidase. (h) an auxin enzyme
or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A
carboxylase (ACCase).
28. An expression cassette comprising a nucleic acid molecule
operably linked to a heterologous promoter, wherein said
heterologous promoter drives expression in a plant and wherein said
nucleic acid molecule is selected from the group consisting of:
a) a nucleic acid molecule comprising the nucleotide sequence of
SEQ ID NO: 60;
b) a nucleic acid molecule comprising a nucleotide sequence having
at least 90% sequence identity to the nucleotide sequence of SEQ ID
NO: 60, wherein said nucleotide sequence encodes a polypeptide that
has HPPD activity and is transported into the chloroplast;
c) a nucleic acid molecule that encodes a polypeptide comprising
the amino acid sequence of SEQ ID NO: 57; and,
d) a nucleic acid molecule that encodes a polypeptide comprising an
amino acid sequence having at least 90% sequence identity to the
amino acid sequence of SEQ ID NO: 57, wherein said nucleotide
sequence encodes a polypeptide that has HPPD activity and is
transported into the chloroplast; and,
e) a complement of any of a)-d).
29. A plant cell comprising at least one expression cassette of
embodiment 28.
30. The plant cell of embodiment 29, wherein said plant cell is a
monocot.
31. The plant cell of embodiment 30, wherein said monocot is maize,
wheat, rice, barley, sorghum, or rye.
32. The plant cell of embodiment 30, wherein said plant is from a
dicot.
33. The plant cell of embodiment 32, wherein said dicot is soybean,
Brassica, sunflower, cotton, or alfalfa.
34. A plant comprising at least one plant cell of any one of
embodiments 29-33.
35. A transgenic seed produced by the plant of embodiment 34,
wherein the seed comprises said expression cassette.
36. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID
NO:57; or, b) a polypeptide comprising an amino acid sequence
having at least 90% sequence identity to the amino acid sequence of
SEQ ID NO:57, wherein said polypeptide has HPPD activity and is
transported into the chloroplast of a plant cell.
37. The plant, plant cell, or seed of any one of embodiments 29-35,
wherein the plant, plant cell, or seed further comprises at least
one polypeptide imparting tolerance to an additional herbicide.
38. The plant, plant cell, or seed of embodiment 37, wherein said
at least one polypeptide imparting tolerance to an additional
herbicide comprises: (a) a sulfonylurea-tolerant acetolactate
synthase; (b) an imidazolinone-tolerant acetolactate synthase; (c)
a glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase;
(d) a glyphosate-tolerant glyphosate oxido-reductase; (e) a
glyphosate-N-acetyltransferase; (f) a phosphinothricin acetyl
transferase; (g) a protoporphyrinogen oxidase. (h) an auxin enzyme
or receptor; (i) a P450 polypeptide; or, (j) an acetyl coenzyme A
carboxylase (ACCase).
39. The plant, plant cell, or seed of embodiment 37, wherein said
at least one polypeptide imparting tolerance to an additional
herbicide comprises a high resistance allele of acetolactate
synthase (HRA) and/or a glyphosate-N-acetyltransferase
polypeptide.
EXPERIMENTAL
Example 1
Maize HPPD has a Chloroplast Targeting Sequence
Bioinformatic Analysis of Maize HPPD:
Maize HPPD proteins are not predicted to have a chloroplast
targeting peptide N-terminal sequence by ProtComp 6.1
(http://linux1.softberry.com/berry.phtml), a widely used program
for detecting organellar targeting sequences. ProtComp 6.1
indicates a cytosolic location of maize HPPD. The results returned
by the search are as follows:
Significant similarity in Location: Cytoplasmic
Cytoplasmic score=14470
Chloroplastic score=1.4
Similarly, WoLF PSORT (Horton et al. (2007) NAR 35:W585-W587) and
TargetP (Emanuelsson et al., (2000) J. Mol. Biol. 300:1005-1016)
predict a cytosolic location of the HPPD protein. Protein Prowler
(Hawkins and Boden (2006) J> Bioinf. Comp. Bio. 4:1-18) predicts
either a mitochondrial (0.34) or chloroplast (0.39) location and
Multiloc (Hoglund et. al. (2006) Bioinformatics 22:1158-1165)
predicts an extracellular (0.74) localization with the first 50
amino acids of maize HPPD but a chloroplast localization (0.97) for
the full maize HPPD sequence (SEQ ID NO 10). However, MultiLoc
fails to predict a CTP function for the first N-terminal 50 amino
acids of maize HPPD, suggesting that additional sequences may be
important for full function.
Evaluation of the Glycine max HPPD protein (SEQ ID NO: 23) gave
similarly variable results with the various prediction programs
with predictions of peroxisomal, cytoplasmic, extracellular and
chloroplast localization.
Example 2
Activity of Truncated Forms of Maize HPPD
Organellar targeting sequences are usually cleaved after the
peptide enters the organelle. Previous investigators (Fritze I et
al. (2004) Plant Physiology 134:1388-1400; Yang C et al. (2004)
Biochemistry 43: 10414-10423) have shown that native mature maize
HPPD begins at either ala17 or ala23. Variants of the wild-type
maize HPPD protein coding region were created with various lengths
of the amino terminus removed. The sequences were expressed in E.
coli and tested for activity and stability. In each case a
methionine start codon was added to the truncated sequence.
Proteins were designated by the position of their N-terminal amino
acid (all alanines) as in SEQ ID NO: 9. All N-terminal truncated
proteins retained the HPPD activity. Differences in the measured
k.sub.cat may not be significantly different as only a single
measurement was taken for this experiment. Assaying HPPD activity
was carried out as described in Example 1 of Provisional
Application 61/401,456, filed Aug. 13, 2010, Compositions and
Methods Comprising Sequences having Hydroxyphenylpyruvate
Dioxygenase (HPPD) Activity, herein incorporated by reference in it
entirety.
TABLE-US-00003 TABLE 1a Activity of N-terminal truncated- variants
of maize wild-type HPPD. Truncation kcat, min-1 Maize wt 166 Ala12
230 Ala15 177 Ala17 180 Ala20 128 Ala23 184
Replicated data with two shuffled variants clearly showed that when
the proteins were truncated such that their second amino acid
(after the N-terminal methionine) is ala20, no significant
differences in kinetic parameters were found.
TABLE-US-00004 TABLE 1b Kinetic parameters of variants truncated to
ala20 Km, mM kcat, min-1 kcat/Km Full-length var A 6.61 .+-. 0.84
247 .+-. 47.1 37.2 .+-. 2.39 Truncated var. A 6.86 .+-. 0.37 206
.+-. 20.5 30.2 .+-. 4.64 Full-length var. B 11.80 .+-. 0.99 106
.+-. 12.4 9.00 .+-. 0.29 Truncated var. B 11.38 .+-. 0.96 93.3 .+-.
5.41 8.22 .+-. 0.36
To test stability, the variants were heated at various temperatures
in the range of 20.degree. C. to 54.degree. C. for 30 minutes. The
remaining activity was determined by the coupled assay described in
Example 1 of Provisional Application 61/401,456, filed Aug. 13,
2010, Compositions and Methods Comprising Sequences having
Hydroxyphenylpyruvate Dioxygenase (HPPD) Activity, herein
incorporated by reference in it entirety. All variants were stable
at 20.degree. C., but activity declined with incubation
temperatures over 30.degree. C. to nearly nil at 54.degree. C.
There were no differences in stability among wild-type and all
truncated variants. Thus, maize HPPD does not require the
N-terminal region of the protein for full enzymatic function in
vitro.
Example 3
The N-Terminus of Maize HPPD Fused to DsRed is Targeted to
Chloroplasts when Transiently Expressed in Maize Leaf
A vector was constructed in which the portion of the maize HPPD
(SEQ ID NO: 9) gene coding for the N-terminal 50 amino acids was
fused to the gene coding for Discosoma sp. red fluorescence protein
2 (DsRed2) and inserted into a binary expression vector under
control of the maize Rubisco activase promoter (Liu et al. (1996)
Plant Physiol. 112(1): 43-51) and terminated with the Solanum
tuberosum proteinase inhibitor II (pinII) terminator region (An et
al. (1989) Plant Cell 1:115-122) with a hygromycin selection
cassette. Both genes are between left and right border sequences
from Agrobacterium.
A positive control vector was identical to the vector having the
N-terminal 50 amino acids of maize HPPD described above except that
the HPPD CTP was removed and the DsRed2 insert was fused to the
chloroplast targeting peptide of Zea maize rubisco activase, while
a negative control was DsRed2 with no targeting sequence. The
plasmids were transformed into Agrobacterium tumefaciens AGL-1 and
Agro-infiltration was used to introduce the constructs into plant
cells. Agro-infiltration is a well described method (Kapila et. al.
(1997) Plant Science, 122:101-108) of introducing an Agrobacterium
cell suspension to plant cells of intact tissues so that
reproducible infection and subsequent plant derived trans-gene
expression may be measured or studied.
Leaves of 3-week old maize seedlings were infiltrated with the
Agrobacterium, and examined by fluorescence microscopy two days
later (Nikon Eclipse 80i, DsRed filter set). With the vector where
DsRed2 was fused to Rubisco activase CTP, the red fluorescence was
seen in discrete packets in a pattern resembling peri-nuclear
chloroplasts, as expected. A similar pattern was seen when DsRed2
was fused to the N-terminal 50 amino acids of maize HPPD. Without
targeting, fluorescence was diffuse with some concentration in the
nucleus. See FIG. 5.
In another experiment, maize leaf tissue was co-bombarded with DNA
from both the DsRed-containing test plasmids and a plasmid encoding
untargeted cycle 3 green fluorescence protein (C3GFP) using the
PDS-1000 He biolistic particle delivery system (Bio-Rad, Hercules
Calif.). Initial examination was conducted at approximately 24 h
post-bombardment with a Lumar fluorescence stereomicroscope (Carl
Zeiss Inc., Thornwood N.Y.) equipped with both a UV-exciting (Zeiss
Set 01) and red-emitting (Zeiss Set 43 HE) filter set to image the
C3GFP and the DsRed2, respectively. C3GFP fluorescence was captured
using a 488 nm argon laser for excitation and a 500-550 nm band
pass emission filter. DsRed fluorescence was imaged using a 561 nm
diode laser for excitation and a 575-615 nm band pass emission
filter. Chlorophyll fluorescence was captured by combining 561 nm
excitation and a 650-710 nm band pass emission filter.
The majority of maize leaf cells transformed were epidermal cells
but because of the relatively low chlorophyll content of epidermal
plastids it was difficult to verify plastid targeting of DsRed
based on chlorophyll co-localization. Guard cell plastids, however,
contained sufficient chlorophyll to be imaged via chlorophyll
autofluorescence. Moderate to low-expressing guard cell pairs were
chosen to illustrate plastid targeting (FIG. 6).
Transformation with vectors encoding RCA CTP-DsRed and the
N-terminal 50 amino acids of maize HPPD fused to DsRed2 resulted in
plastid targeting of the DsRed reporter. When fused to the known
chloroplast targeting sequence of Rubisco activase, DsRed
co-localized with chlorophyll autofluorescence (FIGS. 6B and 6C),
whereas untargeted C3CFP showed no overlap with the Ds Red signal
(FIG. 6A). Guard cell plastids could also be discerned by the
exclusion of the C3GFP signal (FIGS. 6A, 6D, 6E, 6G and 6J) or the
untargeted DsRed signal (FIGS. 6H and 6I). Plastid targeting of
DsRed linked to the N-terminal 50 amino acids of maize HPPD was
evident by a lack of overlap between the cytosolic C3GFP signal and
the DsRed signal (Figs. F and G).
Example 4
0, 17, 20, 23, 30, 40 and 60 Amino Acid Fusions of Zea mays HPPD
N-Terminal Region to Ds-Red and Visualization of Red Fluorescence
in the Chloroplast
Vectors are constructed in which the portion of the maize HPPD gene
(SEQ ID NO: 10) coding for the N-terminal 0, 17, 20, 23, 30, 40 or
60 amino acids are fused to the gene coding for Discosoma sp. red
fluorescence protein 2 (DsRed2) and inserted into a binary
expression vector under control of the maize Rubisco activase
promoter (Liu et al. (1996) Plant Physiol. 112(1): 43-51) and
terminated with the Solanum tuberosum proteinase inhibitor II
(pinII) terminator region (An et al. (1989) Plant Cell 1:115-122)
with a hygromycin selection cassette. Both genes are between left
and right border sequences from Agrobacterium.
A positive control vector is identical except that the insert was
DsRed2 fused to the chloroplast targeting peptide of Arabidopsis
rubisco activase, while a negative control is DsRed2 with no
targeting sequence. The plasmids are transformed into Agrobacterium
tumefaciens AGL-1 and Agro-infiltration used to introduce the
constructs into plant cells. Agro-infiltration is a well described
method (Kapila, et. al., (1997) Plant Science, 122:101-108) of
introducing an Agrobacterium cell suspension to plant cells of
intact tissues so that reproducible infection and subsequent plant
derived trans-gene expression may be measured or studied.
Leaves of 3-week old maize seedlings are infiltrated with the
Agrobacterium, and examined by fluorescence microscopy two days
later (Nikon Eclipse 80i, DsRed filter set).
Example 5
Alignment of Monocot N-Terminal Regions to Show Similarity
FIG. 4 provides an N terminal alignment of monocot HPPD proteins
with identities highlighted. The % identity table shows the
relatedness of the fragments as shown. The proposed CTP activity is
expected to be in the first 17-30 amino acids of each, although the
sequences beyond the cleaved fragments may be important for
localization. In view of the sequence of the monocot HPPD proteins,
a consensus monocot HPPD chloroplast targeting peptide sequences
was determined and provided in SEQ ID NO:1, where the * represents
gaps in the alignment such that those position may be absent in a
variant of the consensus sequence SEQ ID NO 2.
TABLE-US-00005 (SEQ ID NO: 1)
MPPTP(T/A)(T/P/A)(T/P/A)(A/T)(G/T/A)(G/T/A)(G/A/*)(A/*)(G/V/*)(A/S/V)AA(A/-
S)
(A/S/V)(T/A)(P/G/*)E(H/N/Q)A(A/G/R)(F/P/R)(R/*)(L/*)(V/*)(G/S/*)(H/F/*)(R/-
H/P) (R/N)(F/M/V)VR(F/A/V)NPRSDRF(H/Q/P)(T/A/V)L(A/S)FHHVE
A synthetic consensus monocot CTP from HPPD is further provided
comprising the sequence set forth in SEQ ID NO: 2.
TABLE-US-00006 (SEQ ID NO: 2)
MPPTPTTAAATGAGAAAAVTPEHAAFRLVGHRRFVRFNPRSDRFHTLAF HHVE.
Example 6
Fusion of Other Monocot N-Terminal Regions to Ds-Red and
Visualization of Red Fluorescence in Maize Chloroplasts
Vectors are constructed in which N-terminal fragments (any amino
acids from 1-20 or 1-60 or any region in between) of monocot HPPD
proteins (SEQ ID NOS 10, 11, 12, 13, 14, 54) and the synthetic
consensus peptide of SEQ ID NO: 2 is fused to the gene coding for
Discosoma sp. red fluorescence protein 2 (DsRed2) and inserted into
a binary expression vector under control of the maize Rubisco
activase promoter (Liu et al. (1996) Plant Physiol. 112(1): 43-51)
and terminated with the Solanum tuberosum proteinase inhibitor II
(pinII) terminator region (An et al. (1989) Plant Cell 1:115-122)
with a hygromycin selection cassette. Both genes are between left
and right border sequences from Agrobacterium.
A positive control vector is identical except that the insert was
DsRed2 fused to the chloroplast targeting peptide of Arabidopsis
rubisco activase, while a negative control is DsRed2 with no
targeting sequence. The plasmids are transformed into Agrobacterium
tumefaciens AGL-1 and Agro-infiltration used to introduce the
constructs into plant cells. Agro-infiltration is a well described
method (Kapila et. al. (1997) Plant Science, 122:101-108) of
introducing an Agrobacterium cell suspension to plant cells of
intact tissues so that reproducible infection and subsequent plant
derived trans-gene expression may be measured or studied.
Leaves of 3-week old maize seedlings are infiltrated with the
Agrobacterium, and examined by fluorescence microscopy two days
later (Nikon Eclipse 80i, DsRed filter set).
Example 7
Recovery of Mature HPPD Protein from Maize Cells and Sequence of
N-Terminus Showing the Cleavage Site after CTP Removal
Purified native HPPD was obtained from maize leaves by affinity
chromatography on a column of immobilized anti-maize HPPD
antibodies. Serum containing anti-maize HPPD antibodies was raised
in rabbits antigenized with recombinant maize wild type
6.times.-his-HPPD produced in E coli and purified by nickel chelate
affinity chromatography. The serum was passed through Protein A
Ceramin Hyper DF to adsorb the IgG fraction. After washing, IgG was
eluted with citrate buffer, pH 2.55, with a yield of 50 mg of IgG
per gram of serum protein. Ten mg of IgG protein were subjected to
the manufacturer's linkage protocol for Affi-Gel Hz (Bio-Rad),
which resulted in the capture of 2 mg of IgG, 20% of which was
anti-maize HPPD.
Maize leaf tissue was frozen in liquid nitrogen and pulverized in a
mortar cooled with liquid nitrogen. The powder was mixed with 5 ml
of 50 mM potassium phosphate, pH 7.3, 100 mM KCl, 10% ethylene
glycol and 2 mM DTT. When thawed, the debris was removed by
screening and the liquid centrifuged at 14,000.times.g, 15 min. The
soluble protein fraction was desalted by passage through a
gel-filtration column equilibrated with 50 mM potassium phosphate,
100 mM KCl, 10% ethylene glycol and the solution passed through the
Affigel-anti-HPPD column. After extensive washing, pure native
maize HPPD was eluted with 2 bed volumes of 0.5 M formic acid, then
immediately neutralized. The preparation was subjected to Edman
sequencing to determine the N-terminal sequence of mature maize
HPPD protein.
Example 8
Function of Maize HPPD N-Terminal Sequence in Localizing Proteins
to the Chloroplasts of Dicot Plant Cells
A vector is constructed in which the portion of the maize HPPD gene
coding for the N-terminal 50 amino acids was fused to the gene
coding for Discosoma sp. red fluorescence protein 2 (DsRed2) and
inserted into a binary expression vector under control of the
Arabidopsis Ubiquitin 10 promoter (Norris, et al. (1993) Plant Mol.
Biol. 21, 895-906) and terminated with the Solanum tuberosum
proteinase inhibitor II (pinII) terminator region (An et al. (1989)
Plant Cell 1:115-122) with a hygromycin selection cassette. Both
genes are between left and right border sequences from
Agrobacterium.
Leaf tissue of bush bean (common bean, Phaseolus vulgaris), are
agro-infiltrated with Agrobacterium bacterial cell cultures of test
and control strains. Infiltrated leaf samples are derived from
plants of uniform developmental stage grown under the same
conditions. Protoplasts are made from the infiltrated leaves 2-3
days after infection. Protoplasts can be isolated with proper
osmoticum and enzyme digestion as by the method described by Rao
and Prakash (1995) J. Biosci. 20:645-655. Protoplasts are examined
using fluorescence microscopy using a Nikon Eclipse 80i, DsRed
filter set to localize the N-terminal fusion proteins.
Example 9
Localization of Proteins Fused to the N-Terminal Fragments of Dicot
HPPD Proteins
Vectors are constructed in which N-terminal fragments (20-60 amino
acids) of dicot plant HPPD proteins (SEQ ID NOS 15-24) are fused to
the gene coding for Discosoma sp. red fluorescence protein 2
(DsRed2) and inserted into a binary expression vector under control
of the maize Rubisco activase promoter (Liu et al. (1996) Plant
Physiol. 112(1): 43-51) or the Arabidopsis Ubiquitin 10 promoter
(Norris et al. (1993) Plant Mol. Biol. 21, 895-906) and terminated
with the Solanum tuberosum proteinase inhibitor II (pinII)
terminator region (An et al. (1989) Plant Cell 1:115-122) with a
hygromycin selection cassette. Both genes are between left and
right border sequences from Agrobacterium. Such vectors can be used
for either stable or transient gene expression in plant cells.
A positive control vector is identical except that the insert was
DsRed2 fused to the chloroplast targeting peptide of Arabidopsis
rubisco activase, while a negative control is DsRed2 with no
targeting sequence. The plasmids are transformed into Agrobacterium
tumefaciens AGL-1 and Agro-infiltration used to introduce the
constructs into plant cells. Agro-infiltration is a well described
method (Kapila, et. al., (1997) Plant Science, 122:101-108) of
introducing an Agrobacterium cell suspension to plant cells of
intact tissues so that reproducible infection and subsequent plant
derived trans-gene expression may be measured or studied.
Leaves of 3-week old maize seedlings are infiltrated with the
Agrobacterium, and examined by fluorescence microscopy two days
later (Nikon Eclipse 80i, DsRed filter set).
Leaf tissue of bush bean (common bean, Phaseolus vulgaris), are
agro-infiltrated with Agrobacterium bacterial cell cultures of test
and control strains. Infiltrated leaf samples are derived from
plants of uniform developmental stage grown under the same
conditions. Protoplasts are made from the infiltrated leaves 2-3
days after infection. Protoplasts can be isolated with proper
osmoticum and enzyme digestion as by the method described by Rao
and Prakash (1995) J. Biosci. 20:645-655. Protoplasts are examined
using fluorescence microscopy using a Nikon Eclipse 80i, DsRed
filter set to localize the N-terminal fusion proteins.
Example 10
Transformation and Regeneration of Transgenic Maize Plants
Immature maize embryos from greenhouse donor plants are bombarded
with a plasmid containing an expression cassette comprising a
CTP-encoding sequence disclosed herein operably linked to a
polynucleotide encoding a polypeptide of interest operably linked
to a promoter and the selectable marker gene PAT (Wohlleben et al.
(1988) Gene 70:25-37), which confers resistance to the herbicide
Bialaphos. Alternatively, the selectable marker gene is provided on
a separate plasmid. Transformation is performed as follows. Media
recipes follow below.
Preparation of Target Tissue
The ears are husked and surface sterilized in 30% Clorox bleach
plus 0.5% Micro detergent for 20 minutes, and rinsed two times with
sterile water. The immature embryos are excised and placed embryo
axis side down (scutellum side up), 25 embryos per plate, on 560Y
medium for 4 hours and then aligned within the 2.5 cm target zone
in preparation for bombardment.
A plasmid vector comprising the CTP-encoding sequence operably
linked to a polynucleotide encoding a polypeptide of interest is
made. This plasmid DNA plus plasmid DNA containing a PAT selectable
marker is precipitated onto 1.1 .mu.m (average diameter) tungsten
pellets using a CaCl.sub.2 precipitation procedure as follows: 100
.mu.l prepared tungsten particles in water; 10 .mu.l (1 .mu.g) DNA
in Tris EDTA buffer (1 .mu.g total DNA); 100 .mu.l 2.5 M
CaCl.sub.2; and, 10 .mu.l 0.1 M spermidine.
Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
The sample plates are bombarded at level #4 in a particle gun. All
samples receive a single shot at 650 PSI, with a total of ten
aliquots taken from each tube of prepared particles/DNA.
Following bombardment, the embryos are kept on 560Y medium for 2
days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for the translocation of the polypeptide of interest to the
chloroplast of the plant cell.
Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA
C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times.SIGMA-1511),
0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88
g/l L-proline (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after
bringing to volume with D-I H.sub.2O); and 8.5 mg/l silver nitrate
(added after sterilizing the medium and cooling to room
temperature). Selection medium (560R) comprises 4.0 g/l N6 basal
salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,
and 2.0 mg/l 2,4-D (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after
bringing to volume with D-I H.sub.2O); and 0.85 mg/l silver nitrate
and 3.0 mg/l bialaphos (both added after sterilizing the medium and
cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO
11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic
acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l
glycine brought to volume with polished D-I H.sub.2O) (Murashige
and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol,
0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic
acid (brought to volume with polished D-I H.sub.2O after adjusting
to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with
D-I H.sub.2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l
bialaphos (added after sterilizing the medium and cooling to
60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS
salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100
g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL,
and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O),
0.1 g/1 myo-inositol, and 40.0 g/l sucrose (brought to volume with
polished D-I H.sub.2O after adjusting pH to 5.6); and 6 g/l
bacto-agar (added after bringing to volume with polished D-I
H.sub.2O), sterilized and cooled to 60.degree. C.
Bombardment and Culture Media
Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA
C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000.times.SIGMA-1511),
0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88
g/l L-proline (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite (added after
bringing to volume with D-I H.sub.2O); and 8.5 mg/l silver nitrate
(added after sterilizing the medium and cooling to room
temperature). Selection medium (560R) comprises 4.0 g/l N6 basal
salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,
and 2.0 mg/l 2,4-D (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after
bringing to volume with D-I H.sub.2O); and 0.85 mg/l silver nitrate
and 3.0 mg/l bialaphos (both added after sterilizing the medium and
cooling to room temperature).
Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO
11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic
acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l
glycine brought to volume with polished D-I H.sub.2O) (Murashige
and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol,
0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic
acid (brought to volume with polished D-I H.sub.2O after adjusting
to pH 5.6); 3.0 g/l Gelrite (added after bringing to volume with
D-I H.sub.2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l
bialaphos (added after sterilizing the medium and cooling to
60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS
salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100
g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL,
and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O),
0.1 g/1 myo-inositol, and 40.0 g/l sucrose (brought to volume with
polished D-I H.sub.2O after adjusting pH to 5.6); and 6 g/l
bacto-agar (added after bringing to volume with polished D-I
H.sub.2O), sterilized and cooled to 60.degree. C.
Example 11
Agrobacterium-Mediated Transformation of Maize Plants
For Agrobacterium-mediated transformation of maize with a
CTP-encoding sequence operably linked to a polynucleotide encoding
a polypeptide of interest, the method of Zhao is employed (U.S.
Pat. No. 5,981,840, and PCT patent publication WO98/32326; the
contents of which are hereby incorporated by reference). Briefly,
immature embryos are isolated from maize and the embryos contacted
with a suspension of Agrobacterium, where the bacteria are capable
of transferring a CTP-encoding sequence operably linked to a
polynucleotide encoding a polypeptide of interest to at least one
cell of at least one of the immature embryos (step 1: the infection
step). In this step the immature embryos are immersed in an
Agrobacterium suspension for the initiation of inoculation. The
embryos are co-cultured for a time with the Agrobacterium (step 2:
the co-cultivation step). The immature embryos are cultured on
solid medium following the infection step. Following this
co-cultivation period an optional "resting" step is contemplated.
In this resting step, the embryos are incubated in the presence of
at least one antibiotic known to inhibit the growth of
Agrobacterium without the addition of a selective agent for plant
transformants (step 3: resting step). The immature embryos are
cultured on solid medium with antibiotic, but without a selecting
agent, for elimination of Agrobacterium and for a resting phase for
the infected cells. Next, inoculated embryos are cultured on medium
containing a selective agent and growing transformed callus is
recovered (step 4: the selection step). The immature embryos are
cultured on solid medium with a selective agent resulting in the
selective growth of transformed cells. The callus is then
regenerated into plants (step 5: the regeneration step), and calli
grown on selective medium are cultured on solid medium to
regenerate the plants.
Example 12
Soybean Embryo Stable Transformation
Soybean embryos are transformed with an expression cassette
comprising a CTP-encoding sequence disclosed herein operably linked
to a polynucleotide encoding a polypeptide of interest operably
linked to a promoter and the selectable marker gene. Transformation
is performed as follows.
Soybean embryogenic suspension cultures (cv. Jack) are maintained
in 35 ml liquid medium SB196 (see recipes below) on rotary shaker,
150 rpm, 26.degree. C. with cool white fluorescent lights on 16:8
hr day/night photoperiod at light intensity of 60-85 .mu.E/m2/s.
Cultures are subcultured every 7 days to two weeks by inoculating
approximately 35 mg of tissue into 35 ml of fresh liquid SB196 (the
preferred subculture interval is every 7 days).
Soybean embryogenic suspension cultures are transformed with the
plasmids and DNA fragments by the method of particle gun
bombardment (Klein et al. (1987) Nature, 327:70).
Soybean Embryogenic Suspension Culture Initiation
Soybean cultures are initiated twice each month with 5-7 days
between each initiation.
Pods with immature seeds from available soybean plants 45-55 days
after planting are picked, removed from their shells and placed
into a sterilized magenta box. The soybean seeds are sterilized by
shaking them for 15 minutes in a 5% Clorox solution with 1 drop of
ivory soap (95 ml of autoclaved distilled water plus 5 ml Clorox
and 1 drop of soap). Mix well. Seeds are rinsed using 2 l-liter
bottles of sterile distilled water and those less than 4 mm are
placed on individual microscope slides. The small end of the seed
are cut and the cotyledons pressed out of the seed coat. Cotyledons
are transferred to plates containing SB1 medium (25-30 cotyledons
per plate). Plates are wrapped with fiber tape and stored for 8
weeks. After this time secondary embryos are cut and placed into
SB196 liquid media for 7 days.
Preparation of DNA for Bombardment
Either an intact plasmid or a DNA plasmid fragment containing the
genes of interest and the selectable marker gene are used for
bombardment. Plasmid DNA for bombardment are routinely prepared and
purified using the method described in the Promega.TM. Protocols
and Applications Guide, Second Edition (page 106). Fragments of the
plasmids carrying a CTP-encoding sequence operably linked to a
polynucleotide encoding a polypeptide of interest are obtained by
gel isolation of double digested plasmids. In each case, 100 ug of
plasmid DNA is digested in 0.5 ml of the specific enzyme mix that
is appropriate for the plasmid of interest. The resulting DNA
fragments are separated by gel electrophoresis on 1% SeaPlaque GTG
agarose (BioWhitaker Molecular Applications) and the DNA fragments
containing a CTP-encoding sequence operably linked to a
polynucleotide encoding a polypeptide of interest are cut from the
agarose gel. DNA is purified from the agarose using the GELase
digesting enzyme following the manufacturer's protocol.
A 50 .mu.l aliquot of sterile distilled water containing 3 mg of
gold particles (3 mg gold) is added to 5 .mu.l of a 1 .mu.g/0 DNA
solution (either intact plasmid or DNA fragment prepared as
described above), 50 .mu.l 2.5M CaCl.sub.2 and 20 .mu.l of 0.1 M
spermidine. The mixture is shaken 3 min on level 3 of a vortex
shaker and spun for 10 sec in a bench microfuge. After a wash with
400 .mu.l 100% ethanol the pellet is suspended by sonication in 40
.mu.l of 100% ethanol. Five .mu.l of DNA suspension is dispensed to
each flying disk of the Biolistic PDS1000/HE instrument disk. Each
5 .mu.l aliquot contains approximately 0.375 mg gold per
bombardment (i.e. per disk).
Tissue Preparation and Bombardment with DNA
Approximately 150-200 mg of 7 day old embryonic suspension cultures
are placed in an empty, sterile 60.times.15 mm petri dish and the
dish covered with plastic mesh. Tissue is bombarded 1 or 2 shots
per plate with membrane rupture pressure set at 1100 PSI and the
chamber evacuated to a vacuum of 27-28 inches of mercury. Tissue is
placed approximately 3.5 inches from the retaining/stopping
screen.
Selection of Transformed Embryos
Transformed embryos were selected either using hygromycin (when the
hygromycin phosphotransferase, HPT, gene was used as the selectable
marker) or chlorsulfuron (when the acetolactate synthase, ALS, gene
was used as the selectable marker).
Hygromycin (HPT) Selection
Following bombardment, the tissue is placed into fresh SB196 media
and cultured as described above. Six days post-bombardment, the
SB196 is exchanged with fresh SB196 containing a selection agent of
30 mg/L hygromycin. The selection media is refreshed weekly. Four
to six weeks post selection, green, transformed tissue may be
observed growing from untransformed, necrotic embryogenic clusters.
Isolated, green tissue is removed and inoculated into multiwell
plates to generate new, clonally propagated, transformed
embryogenic suspension cultures.
Chlorsulfuron (ALS) Selection
Following bombardment, the tissue is divided between 2 flasks with
fresh SB196 media and cultured as described above. Six to seven
days post-bombardment, the SB196 is exchanged with fresh SB196
containing selection agent of 100 ng/ml Chlorsulfuron. The
selection media is refreshed weekly. Four to six weeks post
selection, green, transformed tissue may be observed growing from
untransformed, necrotic embryogenic clusters. Isolated, green
tissue is removed and inoculated into multiwell plates containing
SB196 to generate new, clonally propagated, transformed embryogenic
suspension cultures.
Regeneration of Soybean Somatic Embryos into Plants
In order to obtain whole plants from embryogenic suspension
cultures, the tissue must be regenerated.
Embryo Maturation
Embryos are cultured for 4-6 weeks at 26.degree. C. in SB196 under
cool white fluorescent (Phillips cool white Econowatt F40/CW/RS/EW)
and Agro (Phillips F40 Agro) bulbs (40 watt) on a 16:8 hr
photoperiod with light intensity of 90-120 uE/m2s. After this time
embryo clusters are removed to a solid agar media, SB166, for 1-2
weeks. Clusters are then subcultured to medium SB103 for 3 weeks.
During this period, individual embryos can be removed from the
clusters and screened for the presence of the polypeptide of
interest in the chloroplast. It should be noted that any detectable
phenotype, resulting from the expression of the genes of interest,
could be screened at this stage.
Embryo Desiccation and Germination
Matured individual embryos are desiccated by placing them into an
empty, small petri dish (35.times.10 mm) for approximately 4-7
days. The plates are sealed with fiber tape (creating a small
humidity chamber). Desiccated embryos are planted into SB71-4
medium where they were left to germinate under the same culture
conditions described above. Germinated plantlets are removed from
germination medium and rinsed thoroughly with water and then
planted in Redi-Earth in 24-cell pack tray, covered with clear
plastic dome. After 2 weeks the dome is removed and plants hardened
off for a further week. If plantlets looked hardy they are
transplanted to 10'' pot of Redi-Earth with up to 3 plantlets per
pot. After 10 to 16 weeks, mature seeds are harvested, chipped and
analyzed for proteins.
Media Recipes
TABLE-US-00007 SB 196 - FN Lite liquid proliferation medium (per
liter) - MS FeEDTA - 100x Stock 1 10 ml MS Sulfate - 100x Stock 2
10 ml FN Lite Halides - 100x Stock 3 10 ml FN Lite P,B,Mo - 100x
Stock 4 10 ml B5 vitamins (1 ml/L) 1.0 ml 2,4-D (10 mg/L final
concentration) 1.0 ml KNO.sub.3 2.83 gm (NH4)2 SO 4 0.463 gm
Asparagine 1.0 gm Sucrose (1%) 10 gm pH 5.8
FN Lite Stock Solutions
TABLE-US-00008 Stock # 1000 ml 500 ml 1 MS Fe EDTA 100x Stock
Na.sub.2 EDTA* 3.724 g 1.862 g FeSO.sub.4--7H.sub.2O 2.784 g 1.392
g 2 MS Sulfate 100x stock MgSO.sub.4--7H.sub.2O 37.0 g 18.5 g
MnSO.sub.4--H.sub.2O 1.69 g 0.845 g ZnSO.sub.4--7H.sub.2O 0.86 g
0.43 g CuSO.sub.4--5H.sub.2O 0.0025 g 0.00125 g 3 FN Lite Halides
100x Stock CaCl.sub.2--2H.sub.2O 30.0 g 15.0 g KI 0.083 g 0.0715 g
CoCl.sub.2--6H.sub.2O 0.0025 g 0.00125 g 4 FN Lite P,B,Mo 100x
Stock KH.sub.2PO.sub.4 18.5 g 9.25 g H.sub.3BO.sub.3 0.62 g 0.31 g
Na.sub.2MoO.sub.4--2H.sub.2O 0.025 g 0.0125 g *Add first, dissolve
in dark bottle while stirring
SB1 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 31.5
g sucrose; 2 ml 2,4-D (20 mg/L final concentration); pH 5.7; and, 8
g TC agar.
SB 166 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 60 g
maltose; 750 mg MgCl2 hexahydrate; 5 g activated charcoal; pH 5.7;
and, 2 g gelrite.
SB 103 solid medium (per liter) comprises: 1 pkg. MS salts
(Gibco/BRL-Cat#11117-066); 1 ml B5 vitamins 1000.times. stock; 60 g
maltose; 750 mg MgCl2 hexahydrate; pH 5.7; and, 2 g gelrite.
SB 71-4 solid medium (per liter) comprises: 1 bottle Gamborg's B5
salts w/sucrose (Gibco/BRL-Cat#21153-036); pH 5.7; and, 5 g TC
agar.
2,4-D stock is obtained premade from Phytotech cat#D
295-concentration is 1 mg/ml.
B5 Vitamins Stock (per 100 ml) which is stored in aliquots at -20 C
comprises: 10 g myo-inositol; 100 mg nicotinic acid; 100 mg
pyridoxine HCl; and, 1 g thiamine. If the solution does not
dissolve quickly enough, apply a low level of heat via the hot stir
plate. Chlorsulfuron Stock comprises 1 mg/ml in 0.01 N Ammonium
Hydroxide.
Example 13
Glycine max HPPD has a Chloroplast Targeting Sequence
The G. max HPPD protein has been previously annotated as a 449
amino acid protein with N-terminal sequence MPIPMCNEIQ (SEQ ID
NO:55) (See, U.S. Pat. No. 7,226,745 SEQ ID NO: 36) and as a 443
amino acid protein with N-terminal sequence MCNEIQAQAQ (SEQ ID NO:
56) (Genbank ABQ96868). Our analysis of G. max EST data revealed
that an in-frame N-terminal extension of the previously annotated
coding region exists, adding 41-amino acids to produce a 488 amino
acid full-length HPPD protein (SEQ ID NO:57).
Bioinformatic and in-planta evaluation of the shorter G. max HPPD
sequences did not reveal a chloroplast or other targeting sequence
by prediction or by localization (see Example 1 and Example 9). The
longer N-terminal region was able to direct a fluorescent marker
protein to the chloroplast in dicot cells. See, Example 14
below.
Bioinformatic analysis of the full-length G. max HPPD (SEQ ID NO:
57) revealed that a chloroplast targeting function is predicted.
ProtComp 9.0 (http://linux1.softberry.com) returned the highest
score for a membrane bound chloroplast localization based on the
first 24 amino acids. WoLF P SORT (http://wolfpsort.org) also
predicts a chloroplast location. TargetP
(http://www.cbs.dtu.dk/services/TargetP) however only suggests a
chloroplast localization, giving a higher score to `other`.
Example 14
Transient Expression of Gm HPPD-AcGFP Fusion Proteins
Numerous genes have been found to have two or more in-frame ATGs at
the 5' end. For review, see Small et al. (1998) Plant Molecular
Biology 38: 265-277. Many of such genes are known to have multiple
transcription starts to enable the production of two proteins from
the same gene. Often, the "long" protein contains plastid targeting
signal at the N-terminal while the "short" protein does not.
Appropriate distribution of the "long" and "short" protein variants
between two subcellular compartments is desired for the respective
protein function to be carried out normally. The soy HPPD gene
described here falls into this class of genes. No other HPPD gene
is known to share the same description.
Transient expression experiments indicate that the long HPPD
protein (SEQ ID NO: 57) is imported to chloroplasts, while the
short protein (SEQ ID NO: 59) remains in the cytosol. Plant
expression cassettes were constructed fusing portions of the N
terminus of Gm HPPD to an Aequorea coerulescens green fluorescent
protein 1 (AcGFP1). One fusion contained amino acid residues 1-86
of the long Gm HPPD protein. Another contained residues 1-44 of the
short HPPD protein (this corresponds to residues 42-86 of the long
protein). These cassettes were incorporated into binary vectors
which also contained an untargeted DsRed2 expression cassette and
introduced into A. tumefaciens strain AGL 1 and then used to infect
leaf discs of G. max as described in Example 3 of U.S. Utility
application Ser. No. 13/209,017, now issued U.S. Pat. No.
8,993,837, entitled "Chimeric Promoter and Methods of Use", filed
concurrently herewith and herein incorporated by reference in its
entirety. As shown in FIG. 8, green fluorescence is clearly visible
in the chloroplasts of infected cells when AcGFP is fused to amino
acid residues 1-86 of Gm HPPD. When the fusion is made with
residues 42-86, corresponding to the 44 N-terminus residues of the
short protein, green fluorescence is visible only in the
cytoplasm.
Example 15
The N-Terminal 0, 10, 20, 30, 40 or 50 Amino Acids of Zea mays HPPD
Fused to Ds-Red; Visualization of Red Fluorescence in the Z. Mays
Chloroplast
Vectors were constructed in which the portion of the maize HPPD
chloroplast targeting sequence (SEQ ID NO: 3) coding for the
N-terminal 0, 10, 20, 30, 40 or 50 amino acids was fused to the
gene coding for Discosoma sp. red fluorescence protein 2 (DsRed2)
and inserted into a binary expression vector under control of the
maize Rubisco activase promoter (Liu et al. (1996) Plant Physiol.
112(1): 43-51) and terminated with the Solanum tuberosum proteinase
inhibitor II (pinII) terminator region (An et al. (1989) Plant Cell
1:115-122). The vector also contained an untargeted Zs Green
cassette to provide cytoplasmic contrast and a kanamycin selection
cassette. All three genes were between left and right border
sequences of Agrobacterium T-DNA. A positive control vector was
identical except that the insert was DsRed2 fused to the
chloroplast targeting peptide of maize rubisco activase, while a
negative control was DsRed2 with no targeting sequence. The
plasmids were transformed into Agrobacterium tumefaciens AGL-1 and
Agro-infiltration was used to introduce the constructs into plant
cells. Agro-infiltration is a well described method (Kapila, et.
al., (1997) Plant Science, 122:101-108) of introducing an
Agrobacterium cell suspension to plant cells of intact tissues so
that reproducible infection and subsequent plant derived trans-gene
expression may be measured or studied. Leaves of 4-week old maize
seedlings were infiltrated with the Agrobacterium, and examined by
fluorescence microscopy four days later (Nikon Eclipse 80i, DsRed
filter set).
Microscopy revealed that 50 amino acids of the maize HPPD
N-terminus effectively targeted DsRed to plastids, but 40 amino
acids or fewer failed to do so, with DsRed fluorescence visible
only in the cytoplasm. See FIG. 9.
Example 16
Fusion of Other Monocot N-Terminal Regions to Ds-Red and
Visualization of red fluorescence in maize and sorghum
chloroplasts
A vector was constructed in which the monocot HPPD N-terminal
consensus sequence (53 amino acids SEQ ID NO: 2) was fused to the
gene coding for DsRed2 and assayed as described in Example 4.
Microscopy revealed that the 53 amino acid consensus sequence
effectively targeted DsRed to maize plastids, as did the maize
rubisco activase positive control, but the untargeted negative
control failed to do so, with DsRed fluorescence visible only in
the cytoplasm.
This vector incorporating the 53 amino acid monocot HPPD N terminal
consensus sequence targeting DsRed, and the 0, 10, 20, 30, 40, and
50 amino acid maize HPPD N-terminal (SEQ ID NO: 3) vectors
described in Example 15 were also tested by Agro-infiltration in
sorghum leaves. The results matched those obtained in maize; ie the
53 amino acid consensus sequence and the 50 amino acid maize
sequence efficiently targeted the DsRed reporter protein to
plastids, but shorter fragments failed to do so with red
fluorescence visible only in the cytoplasm.
A vector was constructed in which the Oriza sativa HPPD N-terminal
sequence (SEQ ID NO: 5) was fused to the gene coding for DsRed2 and
assayed as described in example 4. Microscopy revealed that the 53
amino acid rice N-terminal sequence effectively targeted DsRed to
maize plastids, as did the maize rubisco activase positive control,
and the untargeted negative control failed to do so, with DsRed
fluorescence visible only in the cytoplasm.
Example 17
Function of Maize HPPD N-Terminal Sequence in Localizing Proteins
to the Chloroplasts of Dicot Plant Cells
The sequence encoding amino acids 1-50 of the maize HPPD protein
(SEQ ID NO: 3) was fused to a gene encoding Aequorea coerulescens
green fluorescent protein 1 (AcGFP1) and inserted into a binary
expression vector under control of the Arabidopsis Ubiquitin 10
promoter (Norris et al. (1993) Plant Mol. Biol. 21, 895-906) and
terminated with the Glycine max Kunitz trypsin inhibitor 3
terminator region (NCBI accession S45092). Both genes were between
left and right border sequences from Agrobacterium. Such vectors
can be used for either stable or transient gene expression in plant
cells. A positive control vector was identical except that the
AcGFP1 coding region was fused to the 6H1 synthetic chloroplast
targeting peptide (U.S. Pat. No. 7,345,143 SEQ ID NO:1), while a
negative control was AcGFP1 with no targeting sequence. The
plasmids were transformed into Agrobacterium tumefaciens AGL-1 and
Agro-infiltration used to introduce the constructs into plant
cells. Agro-infiltration is a well described method (Kapila, et.
al., (1997) Plant Science, 122:101-108) of introducing an
Agrobacterium cell suspension to plant cells of intact tissues so
that reproducible infection and subsequent plant derived trans-gene
expression may be measured or studied. Infiltrated leaf samples
were derived from plants of uniform developmental stage grown under
the same conditions. Leaves of 4-week old Nicotiana benthamiana,
8-day old Phaseolus vulgaris, and 10-day old Glycine max seedlings
were infiltrated with the Agrobacterium, and examined by
fluorescence microscopy 4 and 5 days later (Nikon Eclipse 80i,
DsRed filter set). The first 50 amino acids of maize HPPD were
sufficient to drive chloroplast import of AcGFP in epidermal cells
of P. vulgaris, although some green fluorescence remained in the
cytoplasm. In N. benthamiana the AcGFP remained in the cytoplasm
with none apparent in the chloroplasts. Results in G. max showed
AcGFP in both plastids and cytoplasm. When similar constructs were
introduced to soybean leaf epidermal cells and examined by confocal
microscopy, green fluorescence was apparent in the cytosol and
chloroplasts of transformed cells, with variable intensity in
chloroplasts (see FIG. 10). This shows that the maize HPPD CTP is
recognized in dicot plant cells.
Example 18
Localization of Z. Mays HPPD Protein in Stably Transformed Soybean
Cells
Polyclonal antibodies were raised in rabbits against recombinant
maize HPPD protein purified by nickel chelate affinity
chromatography. Anti-HPPD antibodies were purified from serum by
affinity chromatography on immobilized maize HPPD, and further
purified by passage through a column of immobilized Rubisco, to
remove a small fraction of antibodies that reacted with both HPPD
and Rubisco. Leaf punches taken form stably transformed soybean
plants expressing a gene encoding the maize HPPD protein (SEQ ID
NO: 10) driven by an SCP1 synthetic promoter (U.S. Pat. No.
6,072,050) were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde
in 100 mM Na phosphate buffer, pH 7.0, for 3 hours at room
temperature, dehydrated by passage through progressively higher
concentrations of ethanol, embedded in LR White resin and cured at
55.degree. C. for 48 hours. Sections (0.9 microns) were transferred
onto Excell Adhesion glass microscope slides (Electron Microscopy
Sciences). Immunolocalization was performed with the primary
antibody being the double-purified anti-maize HPPD (1:200) and the
secondary antibody goat anti-rabbit F(ab') conjugated with gold
particles (Aurion ultrasmall gold). Gold labeling was followed by
silver enhancement (Aurion R-GENT SE-EM). Sections were
counterstained with 4% uranyl acetate (aqueous) followed by
Reynold's lead citrate. Material was analyzed with a YAG detector
for backscatter signal in a Hitachi 54800 scanning electron
microscope. The clarity of the resulting images was enhanced by
performing contrast inversion, using Adobe PhotoShop CS5.
Gold labeling was observed mainly in cytosol and nuclei, but also
in chloroplasts. See, FIG. 11. A small number of particles observed
in other locations including voids are considered to be artifacts.
This is consistent with transient expression experiments showing
that the first 50 amino acids of the Z. mays HPPD N terminus did
target a fluorescent reporter protein to the chloroplasts of G. max
cells (see Example 17).
Example 19
G. max N-Terminus Targets Proteins to the Chloroplast in Maize
Cells
Vectors were constructed in which a portion of the gene coding for
the G. max HPPD N-terminus was fused to a gene encoding Aequorea
coerulescens green fluorescent protein 1 (AcGFP1) and inserted into
a binary expression vector under control of the Arabidopsis
Ubiquitin 10 promoter (Norris et al. (1993) Plant Mol. Biol. 21,
895-906) and terminated with the Glycine max Kunitz trypsin
inhibitor 3 terminator region (NCBI accession S45092). Both genes
were between left and right border sequences from Agrobacterium.
One fusion contained amino acid residues 1-86 of the long Gm HPPD
protein (SEQ ID NO:57). Another contained residues 1-44 of the
short HPPD protein (this corresponds to residues 42-86 of the long
protein and SEQ ID NO:59).
A positive control vector was identical except that the AcGFP1
coding region was fused to the 6H1 synthetic chloroplast targeting
peptide (U.S. Pat. No. 7,345,143), while a negative control was
AcGFP1 with no targeting sequence. The plasmids were transformed
into Agrobacterium tumefaciens AGL-1 and Agro-infiltration used to
introduce the constructs into plant cells. Agro-infiltration is a
well described method (Kapila et. al. (1997) Plant Science,
122:101-108) of introducing an Agrobacterium cell suspension to
plant cells of intact tissues so that reproducible infection and
subsequent plant trans-gene expression may be measured or
studied.
Leaves of 4-week old maize seedlings were infiltrated with the
Agrobacterium, and examined by fluorescence microscopy three days
later (Nikon Eclipse 80i, Narrow band-pass GFP filter set).
Transient expression indicated that the long G. max HPPD protein
N-terminus (SEQ ID NO: 58) did target the marker protein to maize
cell chloroplasts, while the short protein N-terminus (amino acids
1-44 of SEQ ID NO: 59) delivered the protein to the cytosol. The
dicot chloroplast targeting region of G. max HPPD is able to
function in monocot cells.
TABLE-US-00009 TABLE 2 Summary of SEQ ID NOS SEQ ID NO Description
1 Consensus Monocot CTP from HPPD 2 Synthetic consensus CTP from
monocot HPPD 3 CTP of Zea mays HPPD 4 CTP of sorghum bicolor HPPD 5
CTP of Oryza sativa HPPD 6 CTP of Triticum aestivum HPPD 7 CTP of
Hordeum vulgare HPPD 8 CTP of Avena Sativa HPPD 9 Full length Zea
mays HPPD 10 Maize WT HPPD (from WO1997049816 SEQ ID NO: 11) 11
HPPD from Hordeum vulgare 12 HPPD from Avena sativa 13 HPPD from
Oryza sativa 14 HPPD from Triticum aestivum 15 HPPD from Daucus
carota 16 HPPD from Solenosteman sautellarioides 17 HPPD from Picea
sitchensis 18 HPPD from Abutilon theophrasti 19 HPPD from
Arabidopsis thaliana 20 HPPD from Brassica rapa 21 HPPD from Coptis
japonica 22 HPPD from Vitis vinifera 23 HPPD from Glycine max 24
HPPD from Medicago truncatula 26-53 N-terminal regions of various
HPPD polypeptides 54 HPPD from Sorghum bicolor 55 N-term amino
acids of soy HPPD disclosed in U.S. Pat. No. 7,226,745 as SEQ ID
NO: 36 56 N-term amino acids of soy HPPD disclosed in Genbank
ABQ96868 57 Full length soybean HPPD 58 N-terminal region of SEQ ID
NO: 57 comprising native soybean CTP 59 Soybean HPPD protein
predicted from shorter transcript 60 Nucleotide sequence of the
Full length soybean HPPD
The article "a" and "an" are used herein to refer to one or more
than one (i.e., to at least one) of the grammatical object of the
article. By way of example, "an element" means one or more
element.
All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
SEQUENCE LISTINGS
1
60153PRTArtificial Sequenceconsensus sequence for monocot HPPD CTP
1Met Pro Pro Thr Pro Thr Thr Thr Ala Gly Gly Gly Ala Gly Ala Ala 1
5 10 15Ala Ala Ala Thr Pro Glu His Ala Ala Phe Arg Leu Val Gly His
Arg 20 25 30Arg Phe Val Arg Phe Asn Pro Arg Ser Asp Arg Phe His Thr
Leu Ala 35 40 45Phe His His Val Glu 50253PRTArtificial
Sequenceconsensus sequence for monocot HPPD CTP 2Met Pro Pro Thr
Pro Thr Thr Ala Ala Ala Thr Gly Ala Gly Ala Ala 1 5 10 15Ala Ala
Val Thr Pro Glu His Ala Ala Phe Arg Leu Val Gly His Arg 20 25 30Arg
Phe Val Arg Phe Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ala 35 40
45Phe His His Val Glu 50353PRTZea mays 3Met Pro Pro Thr Pro Thr Ala
Ala Ala Ala Gly Ala Ala Val Ala Ala 1 5 10 15Ala Ser Ala Ala Glu
Gln Ala Ala Phe Arg Leu Val Gly His Arg Asn 20 25 30Phe Val Arg Phe
Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ala Phe 35 40 45His His Val
Glu Leu 50499PRTsorghum bicolor 4Met Pro Pro Thr Pro Thr Thr Ala
Ala Ala Thr Gly Ala Ala Val Ala 1 5 10 15Ala Ala Ser Ala Glu Gln
Ala Ala Phe Arg Leu Val Gly His Arg Asn 20 25 30Phe Val Arg Val Asn
Pro Arg Ser Asp Arg Phe His Thr Leu Ala Phe 35 40 45His His Val Glu
Leu Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg 50 55 60Phe Ser Phe
Gly Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser65 70 75 80Thr
Gly Asn Thr Ala His Ala Ser Leu Leu Leu Arg Ser Gly Ala Leu 85 90
95Ala Phe Leu553PRTOryza sativa 5Met Pro Pro Thr Pro Thr Pro Thr
Ala Thr Thr Gly Ala Val Ser Ala 1 5 10 15Ala Ala Ala Ala Gly Glu
Asn Ala Gly Phe Arg Leu Val Gly His Arg 20 25 30Arg Phe Val Arg Ala
Asn Pro Arg Ser Asp Arg Phe Gln Ala Leu Ala 35 40 45Phe His His Val
Glu 50648PRTTriticum aestivum 6Met Pro Pro Thr Pro Thr Thr Pro Ala
Ala Thr Gly Ala Gly Ala Ala 1 5 10 15Ala Ala Val Thr Pro Glu His
Ala Arg Pro Arg Arg Met Val Arg Phe 20 25 30Asn Pro Arg Ser Asp Arg
Phe His Thr Leu Ser Phe His His Val Glu 35 40 45746PRTHordeum
vulgare 7Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala
Ala Ala 1 5 10 15Val Thr Pro Glu His Ala Arg Pro His Arg Met Val
Arg Phe Asn Pro 20 25 30Arg Ser Asp Arg Phe His Thr Leu Ser Phe His
His Val Glu 35 40 45847PRTAvena Sativa 8Met Pro Pro Thr Pro Ala Thr
Ala Thr Gly Ala Ala Ala Ala Ala Val 1 5 10 15Thr Pro Glu His Ala
Ala Arg Ser Phe Pro Arg Val Val Arg Val Asn 20 25 30Pro Arg Ser Asp
Arg Phe Pro Val Leu Ser Phe His His Val Glu 35 40 459444PRTZea mays
9Met Gly Pro Thr Pro Thr Ala Ala Ala Ala Gly Ala Ala Val Ala Ala 1
5 10 15Ala Ser Ala Ala Glu Gln Ala Ala Phe Arg Leu Val Gly His Arg
Asn 20 25 30Phe Val Arg Phe Asn Pro Arg Ser Asp Arg Phe His Thr Leu
Ala Phe 35 40 45His His Val Glu Leu Trp Cys Ala Asp Ala Ala Ser Ala
Ala Gly Arg 50 55 60Phe Ser Phe Gly Leu Gly Ala Pro Leu Ala Ala Arg
Ser Asp Leu Ser65 70 75 80Thr Gly Asn Ser Ala His Ala Ser Leu Leu
Leu Arg Ser Gly Ser Leu 85 90 95Ser Phe Leu Phe Thr Ala Pro Tyr Ala
His Gly Ala Asp Ala Ala Thr 100 105 110Ala Ala Leu Pro Ser Phe Ser
Ala Ala Ala Ala Arg Arg Phe Ala Ala 115 120 125Asp His Gly Leu Ala
Val Arg Ala Val Ala Leu Arg Val Ala Asp Ala 130 135 140Glu Asp Ala
Phe Arg Ala Ser Val Ala Ala Gly Ala Arg Pro Ala Phe145 150 155
160Gly Pro Val Asp Leu Gly Arg Gly Phe Arg Leu Ala Glu Val Glu Leu
165 170 175Tyr Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Pro Asp Gly
Ala Ala 180 185 190Gly Glu Pro Phe Leu Pro Gly Phe Glu Gly Val Ala
Ser Pro Gly Ala 195 200 205Ala Asp Tyr Gly Leu Ser Arg Phe Asp His
Ile Val Gly Asn Val Pro 210 215 220Glu Leu Ala Pro Ala Ala Ala Tyr
Phe Ala Gly Phe Thr Gly Phe His225 230 235 240Glu Phe Ala Glu Phe
Thr Thr Glu Asp Val Gly Thr Ala Glu Ser Gly 245 250 255Leu Asn Ser
Met Val Leu Ala Asn Asn Ser Glu Asn Val Leu Leu Pro 260 265 270Leu
Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Gln Ile Gln Thr 275 280
285Phe Leu Asp His His Gly Gly Pro Gly Val Gln His Met Ala Leu Ala
290 295 300Ser Asp Asp Val Leu Arg Thr Leu Arg Glu Met Gln Ala Arg
Ser Ala305 310 315 320Met Gly Gly Phe Glu Phe Met Ala Pro Pro Thr
Ser Asp Tyr Tyr Asp 325 330 335Gly Val Arg Arg Arg Ala Gly Asp Val
Leu Thr Glu Ala Gln Ile Lys 340 345 350Glu Cys Gln Glu Leu Gly Val
Leu Val Asp Arg Asp Asp Gln Gly Val 355 360 365Leu Leu Gln Ile Phe
Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe 370 375 380Leu Glu Ile
Ile Gln Arg Ile Gly Cys Met Glu Lys Asp Glu Lys Gly385 390 395
400Gln Glu Tyr Gln Lys Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe
405 410 415Ser Gln Leu Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Leu
Glu Ala 420 425 430Lys Gln Ala Ala Ala Ala Ala Ala Ala Gln Gly Ser
435 44010444PRTZea mays 10Met Pro Pro Thr Pro Thr Ala Ala Ala Ala
Gly Ala Ala Val Ala Ala1 5 10 15Ala Ser Ala Ala Glu Gln Ala Ala Phe
Arg Leu Val Gly His Arg Asn 20 25 30Phe Val Arg Phe Asn Pro Arg Ser
Asp Arg Phe His Thr Leu Ala Phe 35 40 45His His Val Glu Leu Trp Cys
Ala Asp Ala Ala Ser Ala Ala Gly Arg 50 55 60Phe Ser Phe Gly Leu Gly
Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser65 70 75 80Thr Gly Asn Ser
Ala His Ala Ser Leu Leu Leu Arg Ser Gly Ser Leu 85 90 95Ser Phe Leu
Phe Thr Ala Pro Tyr Ala His Gly Ala Asp Ala Ala Thr 100 105 110Ala
Ala Leu Pro Ser Phe Ser Ala Ala Ala Ala Arg Arg Phe Ala Ala 115 120
125Asp His Gly Leu Ala Val Arg Ala Val Ala Leu Arg Val Ala Asp Ala
130 135 140Glu Asp Ala Phe Arg Ala Ser Val Ala Ala Gly Ala Arg Pro
Ala Phe145 150 155 160Gly Pro Val Asp Leu Gly Arg Gly Phe Arg Leu
Ala Glu Val Glu Leu 165 170 175Tyr Gly Asp Val Val Leu Arg Tyr Val
Ser Tyr Pro Asp Gly Ala Ala 180 185 190Gly Glu Pro Phe Leu Pro Gly
Phe Glu Gly Val Ala Ser Pro Gly Ala 195 200 205Ala Asp Tyr Gly Leu
Ser Arg Phe Asp His Ile Val Gly Asn Val Pro 210 215 220Glu Leu Ala
Pro Ala Ala Ala Tyr Phe Ala Gly Phe Thr Gly Phe His225 230 235
240Glu Phe Ala Glu Phe Thr Thr Glu Asp Val Gly Thr Ala Glu Ser Gly
245 250 255Leu Asn Ser Met Val Leu Ala Asn Asn Ser Glu Asn Val Leu
Leu Pro 260 265 270Leu Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser
Gln Ile Gln Thr 275 280 285Phe Leu Asp His His Gly Gly Pro Gly Val
Gln His Met Ala Leu Ala 290 295 300Ser Asp Asp Val Leu Arg Thr Leu
Arg Glu Met Gln Ala Arg Ser Ala305 310 315 320Met Gly Gly Phe Glu
Phe Met Ala Pro Pro Thr Ser Asp Tyr Tyr Asp 325 330 335Gly Val Arg
Arg Arg Ala Gly Asp Val Leu Thr Glu Ala Gln Ile Lys 340 345 350Glu
Cys Gln Glu Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val 355 360
365Leu Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe
370 375 380Leu Glu Ile Ile Gln Arg Ile Gly Cys Met Glu Lys Asp Glu
Lys Gly385 390 395 400Gln Glu Tyr Gln Lys Gly Gly Cys Gly Gly Phe
Gly Lys Gly Asn Phe 405 410 415Ser Gln Leu Phe Lys Ser Ile Glu Asp
Tyr Glu Lys Ser Leu Glu Ala 420 425 430Lys Gln Ala Ala Ala Ala Ala
Ala Ala Gln Gly Ser 435 44011434PRTHordeum vulgare 11Met Pro Pro
Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala1 5 10 15Val Thr
Pro Glu His Ala Arg Pro His Arg Met Val Arg Phe Asn Pro 20 25 30Arg
Ser Asp Arg Phe His Thr Leu Ser Phe His His Val Glu Phe Trp 35 40
45Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly
50 55 60Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala
His65 70 75 80Ala Ser Gln Leu Leu Arg Ser Gly Ser Leu Ala Phe Leu
Phe Thr Ala 85 90 95Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser
Leu Pro Ser Phe 100 105 110Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala
Asp His Gly Ile Ala Val 115 120 125Arg Ser Val Ala Leu Arg Val Ala
Asp Ala Ala Glu Ala Phe Arg Ala 130 135 140Ser Arg Arg Arg Gly Ala
Arg Pro Ala Phe Ala Pro Val Asp Leu Gly145 150 155 160Arg Gly Phe
Ala Phe Ala Glu Val Glu Leu Tyr Gly Asp Val Val Leu 165 170 175Arg
Phe Val Ser His Pro Asp Gly Thr Asp Val Pro Phe Leu Pro Gly 180 185
190Phe Glu Gly Val Thr Asn Pro Asp Ala Val Asp Tyr Gly Leu Thr Arg
195 200 205Phe Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala
Ala Ala 210 215 220Tyr Ile Ala Gly Phe Thr Gly Phe His Glu Phe Ala
Glu Phe Thr Ala225 230 235 240Glu Asp Val Gly Thr Thr Glu Ser Gly
Leu Asn Ser Val Val Leu Ala 245 250 255Asn Asn Ser Glu Gly Val Leu
Leu Pro Leu Asn Glu Pro Val His Gly 260 265 270Thr Lys Arg Arg Ser
Gln Ile Gln Thr Phe Leu Glu His His Gly Gly 275 280 285Pro Gly Val
Gln His Ile Ala Val Ala Ser Ser Asp Val Leu Arg Thr 290 295 300Leu
Arg Lys Met Arg Ala Arg Ser Ala Met Gly Gly Phe Asp Phe Leu305 310
315 320Pro Pro Pro Leu Pro Lys Tyr Tyr Glu Gly Val Arg Arg Leu Ala
Gly 325 330 335Asp Val Leu Ser Glu Ala Gln Ile Lys Glu Cys Gln Glu
Leu Gly Val 340 345 350Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu
Gln Ile Phe Thr Lys 355 360 365Pro Val Gly Asp Arg Pro Thr Leu Phe
Leu Glu Met Ile Gln Arg Ile 370 375 380Gly Cys Met Glu Lys Asp Glu
Arg Gly Glu Glu Tyr Gln Lys Gly Gly385 390 395 400Cys Gly Gly Phe
Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile 405 410 415Glu Asp
Tyr Glu Lys Ser Leu Glu Ala Lys Gln Ser Ala Ala Val Gln 420 425
430Gly Ser 12440PRTAvena sativa 12Met Pro Pro Thr Pro Ala Thr Ala
Thr Gly Ala Ala Ala Ala Ala Val1 5 10 15Thr Pro Glu His Ala Ala Arg
Ser Phe Pro Arg Val Val Arg Val Asn 20 25 30Pro Arg Ser Asp Arg Phe
Pro Val Leu Ser Phe His His Val Glu Leu 35 40 45Trp Cys Ala Asp Ala
Ala Ser Ala Ala Gly Arg Phe Ser Phe Ala Leu 50 55 60Gly Ala Pro Leu
Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala65 70 75 80His Ala
Ser Leu Leu Leu Arg Ser Gly Ala Leu Ala Phe Leu Phe Thr 85 90 95Ala
Pro Tyr Ala Pro Pro Pro Gln Glu Ala Ala Thr Ala Ala Ala Thr 100 105
110Ala Ser Ile Pro Ser Phe Ser Ala Asp Ala Ala Arg Thr Phe Ala Ala
115 120 125Ala His Gly Leu Ala Val Arg Ser Val Gly Val Arg Val Ala
Asp Ala 130 135 140Ala Glu Ala Phe Arg Val Ser Val Ala Gly Gly Ala
Arg Pro Ala Phe145 150 155 160Ala Pro Ala Asp Leu Gly His Gly Phe
Gly Leu Ala Glu Val Glu Leu 165 170 175Tyr Gly Asp Val Val Leu Arg
Phe Val Ser Tyr Pro Asp Glu Thr Asp 180 185 190Leu Pro Phe Leu Pro
Gly Phe Glu Arg Val Ser Ser Pro Gly Ala Val 195 200 205Asp Tyr Gly
Leu Thr Arg Phe Asp His Val Val Gly Asn Val Pro Glu 210 215 220Met
Ala Pro Val Ile Asp Tyr Met Lys Gly Phe Leu Gly Phe His Glu225 230
235 240Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly
Leu 245 250 255Asn Ser Val Val Leu Ala Asn Asn Ser Glu Ala Val Leu
Leu Pro Leu 260 265 270Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser
Gln Ile Gln Thr Tyr 275 280 285Leu Glu Tyr His Gly Gly Pro Gly Val
Gln His Ile Ala Leu Ala Ser 290 295 300Asn Asp Val Leu Arg Thr Leu
Arg Glu Met Arg Ala Arg Thr Pro Met305 310 315 320Gly Gly Phe Glu
Phe Met Ala Pro Pro Gln Ala Lys Tyr Tyr Glu Gly 325 330 335Val Arg
Arg Ile Ala Gly Asp Val Leu Ser Glu Glu Gln Ile Lys Glu 340 345
350Cys Gln Glu Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu
355 360 365Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Phe
Phe Leu 370 375 380Glu Met Ile Gln Arg Ile Gly Cys Met Glu Lys Asp
Glu Val Gly Gln385 390 395 400Glu Tyr Gln Lys Gly Gly Cys Gly Gly
Phe Gly Lys Gly Asn Phe Ser 405 410 415Glu Leu Phe Lys Ser Ile Glu
Asp Tyr Glu Lys Ser Leu Glu Val Lys 420 425 430Gln Ser Val Val Ala
Gln Lys Ser 435 44013446PRTOryza sativa 13Met Pro Pro Thr Pro Thr
Pro Thr Ala Thr Thr Gly Ala Val Ser Ala 1 5 10 15Ala Ala Ala Ala
Gly Glu Asn Ala Gly Phe Arg Leu Val Gly His Arg 20 25 30Arg Phe Val
Arg Ala Asn Pro Arg Ser Asp Arg Phe Gln Ala Leu Ala 35 40 45Phe His
His Val Glu Leu Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly 50 55 60Arg
Phe Ala Phe Ala Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu65 70 75
80Ser Thr Gly Asn Ser Ala His Ala Ser Leu Leu Leu Arg Ser Ala Ser
85 90 95Val Ala Phe Leu Phe Thr Ala Pro Tyr Gly Gly Asp His Gly Val
Gly 100 105 110Ala Asp Ala Ala Thr Thr Ala Ser Ile Pro Ser Phe Ser
Pro Gly Ala 115 120 125Ala Arg Arg Phe Ala Ala Asp His Gly Leu Ala
Val His Ala Val Ala 130 135 140Leu Arg Val Ala Asp Ala Ala Asp Ala
Phe Arg Ala Ser Val Ala Ala145 150 155 160Gly Ala Arg Pro Ala Phe
Gln Pro Ala Asp Leu Gly Gly Gly Phe Gly 165 170 175Leu Ala Glu Val
Glu Leu Tyr Gly Asp Val Val Leu Arg Phe Val Ser 180 185 190His Pro
Asp Gly Ala Asp Ala Pro Phe Leu Pro Gly Phe Glu Gly Val 195 200
205Ser Asn Pro Gly Ala Val Asp Tyr Gly Leu Arg Arg Phe Asp His Val
210 215 220Val Gly Asn Val Pro Glu Leu Ala Pro Val Ala Ala Tyr Ile
Ser
Gly225 230 235 240Phe Thr Gly Phe His Glu Phe Ala Glu Phe Thr Ala
Glu Asp Val Gly 245 250 255Thr Ala Glu Ser Gly Leu Asn Ser Val Val
Leu Ala Asn Asn Ala Glu 260 265 270Thr Val Leu Leu Pro Leu Asn Glu
Pro Val His Gly Thr Lys Arg Arg 275 280 285Ser Gln Ile Gln Thr Tyr
Leu Asp His His Gly Gly Pro Gly Val Gln 290 295 300His Ile Ala Leu
Ala Ser Asp Asp Val Leu Gly Thr Leu Arg Glu Met305 310 315 320Arg
Ala Arg Ser Ala Met Gly Gly Phe Glu Phe Leu Ala Pro Pro Pro 325 330
335Pro Asn Tyr Tyr Asp Gly Val Arg Arg Arg Ala Gly Asp Val Leu Ser
340 345 350Glu Glu Gln Ile Asn Glu Cys Gln Glu Leu Gly Val Leu Val
Asp Arg 355 360 365Asp Asp Gln Gly Val Leu Leu Gln Ile Phe Thr Lys
Pro Val Gly Asp 370 375 380Arg Pro Thr Phe Phe Leu Glu Met Ile Gln
Arg Ile Gly Cys Met Glu385 390 395 400Lys Asp Glu Ser Gly Gln Glu
Tyr Gln Lys Gly Gly Cys Gly Gly Phe 405 410 415Gly Lys Gly Asn Phe
Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu 420 425 430Lys Ser Leu
Glu Ala Lys Gln Ala Pro Thr Val Gln Gly Ser 435 440
44514436PRTTriticum aestivum 14Met Pro Pro Thr Pro Thr Thr Pro Ala
Ala Thr Gly Ala Gly Ala Ala 1 5 10 15Ala Ala Val Thr Pro Glu His
Ala Arg Pro Arg Arg Met Val Arg Phe 20 25 30Asn Pro Arg Ser Asp Arg
Phe His Thr Leu Ser Phe His His Val Glu 35 40 45Phe Trp Cys Ala Asp
Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala 50 55 60Leu Gly Ala Pro
Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser65 70 75 80Val His
Ala Ser Gln Leu Leu Arg Ser Gly Asn Leu Ala Phe Leu Phe 85 90 95Thr
Ala Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro 100 105
110Ser Phe Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Leu
115 120 125Ala Val Arg Ser Ile Ala Leu Arg Val Ala Asp Ala Ala Glu
Ala Phe 130 135 140Arg Ala Ser Val Asp Gly Gly Ala Arg Pro Ala Phe
Ser Pro Val Asp145 150 155 160Leu Gly Arg Gly Phe Gly Phe Ala Glu
Val Glu Leu Tyr Gly Asp Val 165 170 175Val Leu Arg Phe Val Ser His
Pro Asp Asp Thr Asp Val Pro Phe Leu 180 185 190Pro Gly Phe Glu Gly
Val Ser Asn Pro Asp Ala Val Asp Tyr Gly Leu 195 200 205Thr Arg Phe
Asp His Val Val Gly Asn Val Pro Glu Leu Ala Pro Ala 210 215 220Ala
Ala Tyr Val Ala Gly Phe Ala Gly Phe His Glu Phe Ala Glu Phe225 230
235 240Thr Thr Glu Asp Val Gly Thr Ala Glu Ser Gly Leu Asn Ser Met
Val 245 250 255Leu Ala Asn Asn Ser Glu Gly Val Leu Leu Pro Leu Asn
Glu Pro Val 260 265 270His Gly Thr Lys Arg Arg Ser Gln Ile Gln Thr
Phe Leu Glu His His 275 280 285Gly Gly Ser Gly Val Gln His Ile Ala
Val Ala Ser Ser Asp Val Leu 290 295 300Arg Thr Leu Arg Glu Met Arg
Ala Arg Ser Ala Met Gly Gly Phe Asp305 310 315 320Phe Leu Pro Pro
Arg Cys Arg Lys Tyr Tyr Glu Gly Val Arg Arg Ile 325 330 335Ala Gly
Asp Val Leu Ser Glu Ala Gln Ile Lys Glu Cys Gln Glu Leu 340 345
350Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu Leu Gln Ile Phe
355 360 365Thr Lys Pro Val Gly Asp Arg Pro Thr Leu Phe Leu Glu Met
Ile Gln 370 375 380Arg Ile Gly Cys Met Glu Lys Asp Glu Arg Gly Glu
Glu Tyr Gln Lys385 390 395 400Gly Gly Cys Gly Gly Phe Gly Lys Gly
Asn Phe Ser Glu Leu Phe Lys 405 410 415Ser Ile Glu Asp Tyr Glu Lys
Ser Leu Glu Ala Lys Gln Ser Ala Ala 420 425 430Val Gln Gly Ser
43515316PRTDaucus carota 15Met Gly Lys Lys Gln Ser Glu Ala Glu Ile
Leu Ser Ser Asn Ser Ser 1 5 10 15Asn Thr Ser Pro Ala Thr Phe Lys
Leu Val Gly Phe Asn Asn Phe Val 20 25 30Arg Ala Asn Pro Lys Ser Asp
His Phe Ala Val Lys Arg Phe His His 35 40 45Ile Glu Phe Trp Cys Gly
Asp Ala Thr Asn Thr Ser Arg Arg Phe Ser 50 55 60Trp Gly Leu Gly Met
Pro Leu Val Ala Lys Ser Asp Leu Ser Thr Gly65 70 75 80Asn Ser Val
His Ala Ser Tyr Leu Val Arg Ser Ala Asn Leu Ser Phe 85 90 95Val Phe
Thr Ala Pro Tyr Ser Pro Ser Thr Thr Thr Ser Ser Gly Ser 100 105
110Ala Ala Ile Pro Ser Phe Ser Ala Ser Gly Phe His Ser Phe Ala Ala
115 120 125Lys His Gly Leu Ala Val Arg Ala Ile Ala Leu Glu Val Ala
Asp Val 130 135 140Ala Ala Ala Phe Glu Ala Ser Val Ala Arg Gly Ala
Arg Pro Ala Ser145 150 155 160Ala Pro Val Glu Leu Asp Asp Gln Ala
Trp Leu Ala Glu Val Glu Leu 165 170 175Tyr Gly Asp Val Val Leu Arg
Phe Val Ser Phe Gly Arg Glu Glu Gly 180 185 190Leu Phe Leu Pro Gly
Phe Glu Ala Val Glu Gly Thr Ala Ser Phe Pro 195 200 205Asp Leu Asp
Tyr Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val 210 215 220Thr
Glu Leu Gly Pro Val Val Glu Tyr Ile Lys Gly Phe Thr Gly Phe225 230
235 240His Glu Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Leu Glu
Ser 245 250 255Gly Leu Asn Ser Val Val Leu Ala Asn Asn Glu Glu Met
Val Leu Leu 260 265 270Pro Leu Asn Glu Pro Val Tyr Gly Thr Lys Arg
Lys Ser Gln Ile Gln 275 280 285Thr Tyr Leu Glu His Asn Glu Gly Ala
Gly Val Gln His Leu Ala Leu 290 295 300Val Ser Glu Asp Ile Phe Arg
Thr Leu Arg Glu Met305 310 31516436PRTSolenosteman sautellarioides
16Met Gly Gln Glu Ser Thr Ala Ala Ala Ala Val Val Pro Ala Glu Phe 1
5 10 15Lys Leu Val Gly His Lys Asn Phe Val Arg Ser Asn Pro Met Ser
Asp 20 25 30His Phe Pro Val His Arg Phe His His Val Glu Phe Trp Cys
Gly Asp 35 40 45Ala Thr Asn Thr Ser Arg Arg Phe Ser Trp Gly Leu Gly
Met Pro Leu 50 55 60Val Ala Lys Ser Asp Leu Ser Thr Gly Asn Ser Ala
His Ala Ser Tyr65 70 75 80Leu Leu Arg Ser Gly Glu Leu Ser Phe Val
Phe Thr Ala Pro Tyr Ser 85 90 95Pro Ser Leu Ala Glu Pro Ser Ser Ala
Ser Ile Pro Thr Phe Ser Phe 100 105 110Ser Asp His Arg Ala Phe Thr
Ser Ser His Gly Leu Ala Val Arg Ala 115 120 125Val Ala Ile Gln Val
Asp Ser Ala Ser Ser Ala Tyr Ser Ala Ala Val 130 135 140Ser Arg Gly
Ala Lys Pro Val Ser Pro Pro Val Val Leu Ala Asp Cys145 150 155
160Glu Thr Ala Ile Ala Glu Val His Leu Tyr Gly Asp Thr Val Leu Arg
165 170 175Phe Val Ser Cys Gly Ser Gly Ala Asp Gly Trp Phe Leu Pro
Gly Phe 180 185 190Glu Val Val Gly Asp Gly Val Ser Cys Gln Glu Leu
Asp Tyr Gly Ile 195 200 205Arg Arg Leu Asp His Ala Val Gly Asn Val
Pro Lys Leu Glu Pro Val 210 215 220Val Asp Tyr Leu Lys Lys Phe Thr
Gly Phe His Glu Phe Ala Glu Phe225 230 235 240Thr Ala Glu Asp Val
Gly Thr Ala Glu Ser Gly Leu Asn Ser Val Val 245 250 255Leu Ala Asn
Asn Asn Glu Asn Val Leu Phe Pro Leu Asn Glu Pro Val 260 265 270Tyr
Gly Thr Lys Arg Lys Ser Gln Ile Gln Thr Tyr Leu Asp His Asn 275 280
285Glu Gly Ala Gly Val Gln His Leu Ala Leu Ile Thr Glu Asp Ile Phe
290 295 300Arg Thr Leu Arg Glu Met Arg Lys Arg Ser Glu Val Gly Gly
Phe Glu305 310 315 320Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Arg
Asn Leu Lys Ser Arg 325 330 335Ala Gly Asp Val Leu Ser Asp Glu Gln
Ile Glu Glu Cys Glu Lys Leu 340 345 350Gly Ile Leu Ile Asp Arg Asp
Asp Gln Gly Thr Leu Leu Gln Ile Phe 355 360 365Thr Lys Pro Val Gly
Asp Arg Pro Thr Leu Phe Ile Glu Ile Ile Gln 370 375 380Arg Val Gly
Cys Met Met Lys Asp Glu Glu Gly Lys Met Tyr Gln Lys385 390 395
400Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys
405 410 415Ser Ile Glu Glu Tyr Glu Lys Met Leu Glu Ser Lys Leu Val
Thr Lys 420 425 430Thr Ala Met Ala 43517142PRTPicea sitchensis
17Met Ser Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Phe Val Ser 1
5 10 15Lys Asp Gly Phe Glu Gly Pro Phe Leu Pro Asn Tyr Glu Pro Val
Gln 20 25 30Ser Ile Pro Leu Ser Tyr Gly Ile Ile Arg Val Asp His Ala
Val Gly 35 40 45Asn Val Glu Lys Leu Glu Glu Ala Val Glu Tyr Val Ala
Lys Phe Thr 50 55 60Gly Phe His Arg Phe Ala Glu Phe Thr Ala Glu Asp
Val Gly Thr Ala65 70 75 80Glu Ser Gly Leu Asn Ser Met Val Leu Ala
Ser Asn Asn Glu Met Val 85 90 95Leu Leu Pro Met Asn Glu Pro Val Phe
Gly Thr Lys Arg Lys Ser Gln 100 105 110Ile Gln Thr Tyr Leu Glu His
Asn Glu Gly Pro Gly Leu Gln His Leu 115 120 125Ala Leu Ile Cys Ser
Asp Ile Phe Ser Thr Leu Lys Glu Met 130 135 14018363PRTAbutilon
theophrasti 18Cys Thr Asp Ala Thr Asn Ala Ala Cys Arg Phe Ser Trp
Gly Leu Gly 1 5 10 15Met Gln Phe Val Ala Lys Ser Asp Leu Ser Thr
Gly Asn Leu Ser His 20 25 30Ala Ser Tyr Leu Leu Arg Ser Asp His Leu
Ser Leu Leu Phe Thr Ala 35 40 45Pro Tyr Ser Pro Ser Ile Ala Leu Ser
Gln Asn Ile Ser Pro His Ser 50 55 60Thr Ala Ser Ile Pro Ser Phe Asp
His Thr Leu Cys Arg Ser Phe Ser65 70 75 80Ser Ser His Gly Leu Val
Val Arg Ala Ile Ala Leu Glu Val Glu Asp 85 90 95Ser Glu Thr Ala Phe
Ala Thr Ser Ile Ser Asn Gly Ala Leu Pro Ser 100 105 110Ser Pro Pro
Ile Leu Leu Asp Gly Ala Thr Ile Ser Glu Val Lys Leu 115 120 125Tyr
Gly Asp Val Val Leu Arg Tyr Val Ser Tyr Ser Lys Asn Thr Asn 130 135
140Pro His His Phe Leu Pro Gly Phe Glu Lys Val Glu Asp Asn Leu
Ser145 150 155 160Tyr Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His
Ala Val Cys Cys 165 170 175Val Pro Glu Leu Gly Pro Ala Ile Ser Tyr
Val Lys Ser Phe Thr Gly 180 185 190Phe His Asp Leu Ala Glu Phe Thr
Ala Glu Asp Val Gly Thr Ser Glu 195 200 205Ser Gly Leu Asn Ser Val
Ile Leu Ala Asn Asn Asn Glu Met Val Leu 210 215 220Met Pro Ile Ala
Glu Pro Val Tyr Gly Thr Lys Arg Lys Ser Gln Val225 230 235 240Gln
Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Val Gln His Leu Ala 245 250
255Leu Leu Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu Met Arg Lys Arg
260 265 270Ser Phe Val Gly Gly Phe Glu Phe Met Pro Ser Pro Pro Pro
Thr Tyr 275 280 285Tyr Glu Lys Leu Lys Gln Arg Val Gly Asp Ile Leu
Ser Asp Glu Gln 290 295 300Ile Lys Glu Cys Glu Glu Leu Gly Ile Met
Val Asp Arg Asp Asp Gln305 310 315 320Gly Thr Leu Leu Gln Ile Phe
Thr Lys Pro Ile Gly Asp Arg Pro Thr 325 330 335Ile Leu Leu Glu Ile
Ile Gln Arg Ile Gly Cys Met Val Lys Asp Glu 340 345 350Glu Gly Lys
Gln Tyr Gln Lys Gly Gly Cys Gly 355 36019473PRTArabidopsis thaliana
19Met Cys Leu Ser Leu Ala Ser Thr Ala Gln Arg Asn Thr Lys Phe Arg 1
5 10 15Ser Arg Val Leu Val Leu Ala Glu Leu Val Lys Ser Met Gly His
Gln 20 25 30Asn Ala Ala Val Ser Glu Asn Gln Asn His Asp Asp Gly Ala
Ala Ser 35 40 45Ser Pro Gly Phe Lys Leu Val Gly Phe Ser Lys Phe Val
Arg Lys Asn 50 55 60 Pro Lys Ser Asp Lys Phe Lys Val Lys Arg Phe
His His Ile Glu Phe65 70 75 80Trp Cys Gly Asp Ala Thr Asn Val Ala
Arg Arg Phe Ser Trp Gly Leu 85 90 95Gly Met Arg Phe Ser Ala Lys Ser
Asp Leu Ser Thr Gly Asn Met Val 100 105 110His Ala Ser Tyr Leu Leu
Thr Ser Gly Asp Leu Arg Phe Leu Phe Thr 115 120 125Ala Pro Tyr Ser
Pro Ser Leu Ser Ala Gly Glu Ile Lys Pro Thr Thr 130 135 140Thr Ala
Ser Ile Pro Ser Phe Asp His Gly Ser Cys Arg Ser Phe Phe145 150 155
160Ser Ser His Gly Leu Gly Val Arg Ala Val Ala Ile Glu Val Glu Asp
165 170 175Ala Glu Ser Ala Phe Ser Ile Ser Val Ala Asn Gly Ala Ile
Pro Ser 180 185 190Ser Pro Pro Ile Val Leu Asn Glu Ala Val Thr Ile
Ala Glu Val Lys 195 200 205Leu Tyr Gly Asp Val Val Leu Arg Tyr Val
Ser Tyr Lys Ala Glu Asp 210 215 220Thr Glu Lys Ser Glu Phe Leu Pro
Gly Phe Glu Arg Val Glu Asp Ala225 230 235 240Ser Ser Phe Pro Leu
Asp Tyr Gly Ile Arg Arg Leu Asp His Ala Val 245 250 255Gly Asn Val
Pro Glu Leu Gly Pro Ala Leu Thr Tyr Val Ala Gly Phe 260 265 270Thr
Gly Phe His Gln Phe Ala Glu Phe Thr Ala Asn Asp Val Gly Thr 275 280
285Ala Glu Ser Gly Leu Asn Ser Ala Val Leu Ala Ser Asn Asp Glu Met
290 295 300Val Leu Leu Pro Ile Asn Glu Pro Val His Gly Thr Lys Arg
Lys Ser305 310 315 320Gln Ile Gln Thr Tyr Leu Glu His Asn Glu Gly
Ala Gly Leu Gln His 325 330 335Leu Ala Leu Met Ser Glu Asp Ile Phe
Arg Thr Leu Arg Glu Met Arg 340 345 350Lys Arg Ser Ser Ile Gly Gly
Phe Asp Phe Met Pro Ser Pro Pro Pro 355 360 365Thr Tyr Tyr Gln Asn
Leu Lys Lys Arg Val Gly Asp Val Leu Ser Asp 370 375 380Asp Gln Ile
Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp385 390 395
400Asp Gln Gly Thr Leu Leu Gln Ile Phe Thr Lys Pro Leu Gly Asp Arg
405 410 415Pro Thr Ile Phe Ile Glu Ile Ile Gln Arg Val Gly Cys Met
Met Lys 420 425 430Asp Glu Glu Gly Lys Ala Tyr Gln Ser Gly Gly Cys
Gly Gly Phe Gly 435 440 445Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser
Ile Glu Glu Tyr Glu Lys 450 455 460Thr Leu Glu Ala Lys Gln Leu Val
Gly465 47020443PRTBrassica rapa 20Met Gly His Glu Asn Ala Ala Val
Ser Glu Asn Gln His His Asp Asp 1 5 10 15Ala Ala Thr Thr Ser Ala
Ser Pro Gly Phe Lys Leu Val Gly Phe Ser 20 25 30Lys Phe Val Arg Lys
Asn Pro Lys Ser Asp Lys Phe Lys Val Lys Arg 35 40 45Phe His His Ile
Glu Phe Trp Cys Gly Asp Ala Thr Asn Val Ala Arg 50 55 60Arg Phe Ser
Trp Gly Leu Gly Met Arg Phe Ser Ala Lys Ser Asp Leu65
70 75 80Ser Thr Gly Asn Met Val His Ala Ser Tyr Leu Leu Thr Ser Gly
Asp 85 90 95Leu Arg Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Leu Ser
Ala Gly 100 105 110Glu Asn Pro Pro Thr Thr Thr Ala Ser Ile Pro Ser
Phe Asp His Val 115 120 125Thr Tyr Arg Ser Phe Phe Ser Ser His Gly
Leu Gly Val Arg Ala Val 130 135 140Ala Val Glu Val Glu Asp Ala Glu
Ala Ala Phe Ser Ile Ser Val Ser145 150 155 160Asn Gly Ala Val Pro
Ser Ser Pro Pro Ile Val Leu Asn Asp Ala Val 165 170 175Thr Ile Ala
Glu Val Lys Leu Tyr Gly Asp Val Val Leu Arg Tyr Val 180 185 190Ser
Tyr Lys Val Ala Thr Val Phe Leu Pro Arg Phe Glu Thr Val Asp 195 200
205Asp Thr Ser Ser Phe Pro Leu Asp Tyr Gly Ile Arg Arg Leu Asp His
210 215 220Ala Val Gly Asn Val Pro Glu Leu Gly Pro Ala Leu Thr Tyr
Leu Ser225 230 235 240Arg Leu Thr Gly Phe His Gln Phe Ala Glu Phe
Thr Ala Asp Asp Val 245 250 255Gly Thr Ala Glu Ser Gly Leu Asn Ser
Ala Val Leu Ala Asn Asn Asp 260 265 270Glu Thr Val Leu Leu Pro Val
Asn Glu Pro Val His Gly Thr Lys Arg 275 280 285Lys Ser Gln Ile Gln
Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Val 290 295 300Gln His Leu
Ala Leu Met Ser Glu Asp Ile Phe Arg Thr Leu Arg Glu305 310 315
320Met Arg Lys Arg Ser Gly Val Gly Gly Phe Asp Phe Met Pro Ser Pro
325 330 335Pro Pro Thr Tyr Tyr Lys Asn Leu Lys Asn Arg Val Gly Asp
Val Leu 340 345 350Ser Glu Glu Gln Ile Glu Glu Cys Glu Glu Leu Gly
Ile Leu Val Asp 355 360 365Arg Asp Asp Gln Gly Thr Leu Leu Gln Ile
Phe Thr Lys Pro Leu Gly 370 375 380Asp Arg Pro Thr Ile Phe Ile Glu
Ile Ile Gln Arg Ile Gly Cys Met385 390 395 400Lys Lys Asp Glu Glu
Gly Arg Val Tyr Gln Ser Gly Gly Cys Gly Gly 405 410 415Phe Gly Lys
Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr 420 425 430Glu
Lys Thr Leu Glu Ala Lys Gln Leu Val Gly 435 44021430PRTCoptis
japonica 21Met Val Pro Ser Thr Ala Ser Asn Leu Lys Leu Val Gly His
Thr Asn 1 5 10 15Phe Val His Asn Asn Pro Lys Ser Asp Lys Phe His
Val Lys Lys Phe 20 25 30His His Ile Glu Phe Trp Ser Thr Asp Ala Thr
Asn Thr Ala Arg Arg 35 40 45Phe Ser Trp Gly Leu Gly Met Pro Met Val
Ala Lys Ser Asp Leu Ser 50 55 60Thr Gly Asn Met Val His Ala Ser Tyr
Leu Leu Arg Ser Gly Glu Leu65 70 75 80Asn Phe Leu Phe Thr Ala Pro
Tyr Ser Pro Ser Ile Ala Gly Asn Thr 85 90 95Leu Thr His Thr Ala Ser
Ile Pro Thr Tyr Ser His Asn Leu Ala Arg 100 105 110Leu Phe Ala Ser
Thr His Gly Leu Ala Val Arg Ala Ile Ala Ile Glu 115 120 125Val Gln
Asp Ala Glu Leu Ala Tyr Asn Ile Ser Val Ala Asn Gly Ala 130 135
140Lys Pro Ser Ser Ser Pro Ile Lys Leu Asp Glu Gly Val Val Leu
Ser145 150 155 160Glu Ile Gln Leu Tyr Gly Asp Val Val Leu Arg Tyr
Leu Ser Phe Lys 165 170 175Asn Thr Asn Gln Ser Cys Pro Phe Leu Pro
Gly Phe Glu Glu Val Gly 180 185 190Glu Val Ser Ser Ser Arg Gly Leu
Asp Phe Gly Ile Arg Arg Leu Asp 195 200 205His Ala Val Gly Asn Val
Pro Asn Leu Ala Glu Ala Ile Gly Tyr Leu 210 215 220Lys Glu Phe Thr
Gly Phe His Glu Phe Ala Glu Phe Thr Ala Glu Asp225 230 235 240Val
Gly Thr Thr Glu Ser Gly Leu Asn Ser Ile Val Leu Ala Ser Asn 245 250
255Asp Glu Met Val Leu Leu Pro Met Asn Glu Pro Val Tyr Gly Thr Lys
260 265 270Arg Lys Ser Gln Ile Gln Thr Tyr Leu Glu His Asn Glu Gly
Ala Gly 275 280 285Val Gln His Leu Ala Leu Val Ser Glu Asp Ile Phe
Thr Thr Leu Arg 290 295 300Glu Met Arg Arg Arg Ser Gly Val Gly Gly
Phe Glu Phe Met Pro Ser305 310 315 320Pro Pro Pro Thr Tyr Tyr Lys
Asn Leu Lys Asn Arg Ala Gly Asp Val 325 330 335Leu Ser Asp Glu Gln
Ile Lys Glu Cys Glu Glu Leu Gly Ile Leu Val 340 345 350Asp Arg Asp
Ala Gln Gly Thr Leu Leu Gln Ile Phe Thr Lys Pro Val 355 360 365Gly
Asp Arg Pro Thr Ile Phe Val Glu Ile Ile Gln Arg Leu Gly Cys 370 375
380Met Leu Lys Asp Glu Glu Gly Lys Thr Tyr Gln Lys Ala Gly Cys
Gly385 390 395 400Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu Phe Lys
Ser Ile Glu Glu 405 410 415Tyr Glu Lys Thr Leu Glu Ala Lys Ala Asn
Val Val Ala Ala 420 425 43022445PRTVitis vinifera 22Met Gly Lys Gln
Asn Thr Thr Thr Asn Asn Pro Ala Pro Gly Phe Lys 1 5 10 15Leu Val
Gly Phe Ser Asn Phe Leu Arg Thr Asn Pro Met Ser Asp Arg 20 25 30Phe
Gly Val Lys Arg Phe His His Ile Glu Phe Trp Ser Thr Asp Ala 35 40
45Thr Asn Leu Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Pro Ile Val
50 55 60Ala Lys Ser Asp Leu Ser Thr Gly Asn Val Ile His Ala Ser Tyr
Leu65 70 75 80Thr Arg Ser Gly Asp Leu Asn Phe Leu Phe Thr Ala Pro
Tyr Ser Pro 85 90 95Ser Ile Ala Gly Asp Leu Glu Asn Ala Ala Ala Thr
Ala Ser Ile Pro 100 105 110Ser Phe Asp His Ser Ala Cys His Ala Phe
Ala Ala Ser His Gly Leu 115 120 125Gly Val Arg Ala Ile Ala Ile Glu
Val Asp Asp Ala Glu Gly Ala Phe 130 135 140His Thr Ser Val Ala His
Gly Ala Arg Pro Met Ser Pro Pro Val Thr145 150 155 160Met Gly Gly
Ser Val Val Ile Ser Glu Val His Leu Tyr Gly Asp Ala 165 170 175Val
Leu Arg Tyr Val Ser Tyr Lys Asn Pro Asn Pro Asn Ala Thr Ser 180 185
190Asp Pro Ser Ser Trp Phe Leu Pro Gly Phe Glu Ala Val Asp Glu Gly
195 200 205Ser Ser Phe Pro Val Asp Phe Gly Leu Arg Arg Val Asp His
Thr Val 210 215 220Gly Asn Val Pro Lys Leu Ala Pro Val Val Thr Tyr
Leu Lys Gln Phe225 230 235 240Thr Gly Phe His Glu Phe Ala Glu Phe
Thr Ala Glu Asp Val Gly Thr 245 250 255Ser Glu Ser Gly Leu Asn Ser
Val Val Leu Ala Ser Asn Asn Glu Met 260 265 270Val Leu Leu Pro Leu
Asn Glu Pro Val Phe Gly Thr Lys Arg Lys Ser 275 280 285Gln Ile Gln
Thr Tyr Leu Glu His Asn Glu Gly Pro Gly Val Gln His 290 295 300Leu
Ala Leu Met Ser Asp Asp Ile Phe Arg Thr Leu Arg Glu Met Arg305 310
315 320Arg Arg Ser Gly Val Gly Gly Phe Asp Phe Met Pro Ser Pro Pro
Pro 325 330 335Thr Tyr Tyr Arg Asn Val Lys Lys Arg Ala Gly Asp Val
Leu Thr Asp 340 345 350Asp Gln Ile Lys Glu Cys Glu Glu Leu Gly Ile
Leu Val Asp Lys Asp 355 360 365Asp Gln Gly Thr Leu Leu Gln Ile Phe
Thr Lys Pro Leu Gly Asp Arg 370 375 380Pro Thr Ile Phe Ile Glu Ile
Ile Gln Arg Leu Gly Cys Met Val Lys385 390 395 400Asp Asp Glu Gly
Lys Val Ser Gln Lys Gly Gly Cys Gly Gly Phe Gly 405 410 415Lys Gly
Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr Glu Lys 420 425
430Thr Leu Gly Ala Lys Arg Ile Val Asp Pro Ala Pro Val 435 440
44523449PRTGlycine max 23Met Pro Ile Pro Met Cys Asn Glu Ile Gln
Ala Gln Ala Gln Ala Gln 1 5 10 15Ala Gln Ala Gln Pro Gly Phe Lys
Leu Val Gly Phe Lys Asn Phe Val 20 25 30Arg Thr Asn Pro Lys Ser Asp
Arg Phe Gln Val Asn Arg Phe His His 35 40 45Ile Glu Phe Trp Cys Thr
Asp Ala Thr Asn Ala Ser Arg Arg Phe Ser 50 55 60Trp Gly Leu Gly Met
Pro Ile Val Ala Lys Ser Asp Leu Ser Thr Gly65 70 75 80Asn Gln Ile
His Ala Ser Tyr Leu Leu Arg Ser Gly Asp Leu Ser Phe 85 90 95Leu Phe
Ser Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Ser Ser Ala 100 105
110Ala Ser Ser Ala Ser Ile Pro Ser Phe Asp Ala Ala Thr Cys Leu Ala
115 120 125Phe Ala Ala Lys His Gly Phe Gly Val Arg Ala Ile Ala Leu
Glu Val 130 135 140Ala Asp Ala Glu Ala Ala Phe Ser Ala Ser Val Ala
Lys Gly Ala Glu145 150 155 160Pro Ala Ser Pro Pro Val Leu Val Asp
Asp Arg Thr Gly Phe Ala Glu 165 170 175Val Arg Leu Tyr Gly Asp Val
Val Leu Arg Tyr Val Ser Tyr Lys Asp 180 185 190Ala Ala Pro Gln Ala
Pro His Ala Asp Pro Ser Arg Trp Phe Leu Pro 195 200 205Gly Phe Glu
Ala Ala Ala Ser Ser Ser Ser Phe Pro Glu Leu Asp Tyr 210 215 220Gly
Ile Arg Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Ala225 230
235 240Pro Ala Val Arg Tyr Leu Lys Gly Phe Ser Gly Phe His Glu Phe
Ala 245 250 255Glu Phe Thr Ala Glu Asp Val Gly Thr Ser Glu Ser Gly
Leu Asn Ser 260 265 270Val Val Leu Ala Asn Asn Ser Glu Thr Val Leu
Leu Pro Leu Asn Glu 275 280 285Pro Val Tyr Gly Thr Lys Arg Lys Ser
Gln Ile Glu Thr Tyr Leu Glu 290 295 300His Asn Glu Gly Ala Gly Val
Gln His Leu Ala Leu Val Thr His Asp305 310 315 320Ile Phe Thr Thr
Leu Arg Glu Met Arg Lys Arg Ser Phe Leu Gly Gly 325 330 335Phe Glu
Phe Met Pro Ser Pro Pro Pro Thr Tyr Tyr Ala Asn Leu His 340 345
350Asn Arg Ala Ala Asp Val Leu Thr Val Asp Gln Ile Lys Gln Cys Glu
355 360 365Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gln Gly Thr Leu
Leu Gln 370 375 380Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Ile
Phe Ile Glu Ile385 390 395 400Ile Gln Arg Ile Gly Cys Met Val Glu
Asp Glu Glu Gly Lys Val Tyr 405 410 415Gln Lys Gly Ala Cys Gly Gly
Phe Gly Lys Gly Asn Phe Ser Glu Leu 420 425 430Phe Lys Ser Ile Glu
Glu Tyr Glu Lys Thr Leu Glu Ala Lys Arg Thr 435 440 445Ala
24437PRTMedicago truncatula 24Met Ala Ile Glu Thr Glu Thr Gln Thr
Gln Thr Gln Thr Gly Phe Lys 1 5 10 15Leu Val Gly Phe Lys Asn Phe
Val Arg Ala Asn Pro Lys Ser Asp Arg 20 25 30Phe Asn Val Lys Arg Phe
His His Val Glu Phe Trp Cys Thr Asp Ala 35 40 45Thr Asn Thr Ala Arg
Arg Phe Ser His Gly Leu Gly Met Pro Ile Val 50 55 60Ala Lys Ser Asp
Leu Ser Thr Gly Asn Leu Thr His Ala Ser Tyr Leu65 70 75 80Leu Arg
Ser Gly Asp Leu Asn Phe Leu Phe Ser Ala Ala Tyr Ser Pro 85 90 95Ser
Ile Ser Leu Ser Ser Pro Ser Ser Thr Ala Ala Ile Pro Thr Phe 100 105
110Ser Ala Ser Thr Cys Phe Ser Phe Ser Ala Ser His Gly Leu Ala Val
115 120 125Arg Ala Val Ala Val Glu Val Glu Asp Ala Glu Val Ala Phe
Thr Thr 130 135 140Ser Val Asn Leu Gly Ala Ile Pro Ser Ser Pro Pro
Val Ile Leu Glu145 150 155 160Asn Asn Val Lys Leu Ala Glu Val His
Leu Tyr Gly Asp Val Val Leu 165 170 175Arg Tyr Val Ser Tyr Asn Asp
Leu Asn Pro Asn Gln Asn Pro Asn Leu 180 185 190Phe Phe Leu Pro Gly
Phe Glu Arg Val Ser Asp Glu Ser Ser Asn Ser 195 200 205Ser Leu Asp
Phe Gly Ile Arg Arg Leu Asp His Ala Val Gly Asn Val 210 215 220Pro
Glu Leu Ser Ser Ala Val Lys Tyr Val Lys Gln Phe Thr Gly Phe225 230
235 240His Glu Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Ser Glu
Ser 245 250 255Gly Leu Asn Ser Val Val Leu Ala Asn Asn Glu Glu Thr
Val Leu Leu 260 265 270Pro Met Asn Glu Pro Val Tyr Gly Thr Lys Arg
Lys Ser Gln Ile Glu 275 280 285Thr Tyr Leu Glu His Asn Glu Gly Ala
Gly Leu Gln His Leu Ala Leu 290 295 300Met Ser Ala Asp Ile Phe Arg
Thr Leu Arg Glu Met Arg Lys Arg Ser305 310 315 320Gly Val Gly Gly
Phe Glu Phe Met Pro Ser Pro Pro Val Thr Tyr Tyr 325 330 335Arg Asn
Leu Lys Asn Arg Val Gly Asp Val Leu Ser Asp Glu Gln Ile 340 345
350Lys Glu Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp Asp Gln Gly
355 360 365Thr Leu Leu Gln Ile Phe Thr Lys Pro Ile Gly Asp Arg Pro
Thr Ile 370 375 380Phe Ile Glu Ile Ile Gln Arg Val Gly Cys Met Leu
Lys Asp Glu Glu385 390 395 400Gly Lys Glu Tyr Gln Lys Gly Gly Cys
Gly Gly Phe Gly Lys Gly Asn 405 410 415Phe Ser Glu Leu Phe Lys Ser
Ile Glu Glu Tyr Glu Lys Thr Leu Glu 420 425 430Thr Arg Arg Thr Ala
43525152PRTZea mays 25Met Pro Pro Thr Pro Thr Ala Ala Ala Ala Gly
Ala Ala Val Ala Ala 1 5 10 15Ala Ser Ala Ala Glu Gln Ala Ala Phe
Arg Leu Val Gly His Arg Asn 20 25 30Phe Val Arg Phe Asn Pro Arg Ser
Asp Arg Phe His Thr Leu Ala Phe 35 40 45His His Val Glu Leu Trp Cys
Ala Asp Ala Ala Ser Ala Ala Gly Arg 50 55 60Phe Ser Phe Gly Leu Gly
Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser65 70 75 80Thr Gly Asn Ser
Ala His Ala Ser Leu Leu Leu Arg Ser Gly Ser Leu 85 90 95Ser Phe Leu
Phe Thr Ala Pro Tyr Ala His Gly Ala Asp Ala Ala Thr 100 105 110Ala
Ala Leu Pro Ser Phe Ser Ala Ala Ala Ala Arg Arg Phe Ala Ala 115 120
125Asp His Gly Leu Ala Val Arg Ala Val Ala Leu Arg Val Ala Asp Ala
130 135 140Glu Asp Ala Phe Arg Ala Ser Val145 15026158PRTOryza
sativa 26Met Pro Pro Thr Pro Thr Pro Thr Ala Thr Thr Gly Ala Val
Ser Ala 1 5 10 15Ala Ala Ala Ala Gly Glu Asn Ala Gly Phe Arg Leu
Val Gly His Arg 20 25 30Arg Phe Val Arg Ala Asn Pro Arg Ser Asp Arg
Phe Gln Ala Leu Ala 35 40 45Phe His His Val Glu Leu Trp Cys Ala Asp
Ala Ala Ser Ala Ala Gly 50 55 60Arg Phe Ala Phe Ala Leu Gly Ala Pro
Leu Ala Ala Arg Ser Asp Leu65 70 75 80Ser Thr Gly Asn Ser Ala His
Ala Ser Leu Leu Leu Arg Ser Ala Ser 85 90 95Val Ala Phe Leu Phe Thr
Ala Pro Tyr Gly Gly Asp His Gly Val Gly 100 105 110Ala Asp Ala Ala
Thr Thr Ala Ser Ile Pro Ser Phe Ser Pro Gly Ala 115 120 125Ala Arg
Arg Phe Ala Ala Asp His Gly Leu Ala Val His Ala Val Ala 130 135
140Leu Arg Val Ala Asp Ala Ala Asp Ala Phe Arg Ala Ser Val145 150
15527152PRTAvena sativa 27Met Pro Pro Thr Pro Ala Thr Ala Thr Gly
Ala Ala Ala Ala Ala Val 1 5 10
15Thr Pro Glu His Ala Ala Arg Ser Phe Pro Arg Val Val Arg Val Asn
20 25 30Pro Arg Ser Asp Arg Phe Pro Val Leu Ser Phe His His Val Glu
Leu 35 40 45Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe
Ala Leu 50 55 60Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly
Asn Ser Ala65 70 75 80His Ala Ser Leu Leu Leu Arg Ser Gly Ala Leu
Ala Phe Leu Phe Thr 85 90 95Ala Pro Tyr Ala Pro Pro Pro Gln Glu Ala
Ala Thr Ala Ala Ala Thr 100 105 110Ala Ser Ile Pro Ser Phe Ser Ala
Asp Ala Ala Arg Thr Phe Ala Ala 115 120 125Ala His Gly Leu Ala Val
Arg Ser Val Gly Val Arg Val Ala Asp Ala 130 135 140Ala Glu Ala Phe
Arg Val Ser Val145 15028146PRTHordeum vulgare 28Met Pro Pro Thr Pro
Thr Thr Pro Ala Ala Thr Gly Ala Ala Ala Ala 1 5 10 15Val Thr Pro
Glu His Ala Arg Pro His Arg Met Val Arg Phe Asn Pro 20 25 30Arg Ser
Asp Arg Phe His Thr Leu Ser Phe His His Val Glu Phe Trp 35 40 45Cys
Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ala Phe Ala Leu Gly 50 55
60Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser Thr Gly Asn Ser Ala His65
70 75 80Ala Ser Gln Leu Leu Arg Ser Gly Ser Leu Ala Phe Leu Phe Thr
Ala 85 90 95Pro Tyr Ala Asn Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro
Ser Phe 100 105 110Ser Ala Asp Ala Ala Arg Arg Phe Ser Ala Asp His
Gly Ile Ala Val 115 120 125Arg Ser Val Ala Leu Arg Val Ala Asp Ala
Ala Glu Ala Phe Arg Ala 130 135 140Ser Arg14529148PRTTriticum
aestivum 29Met Pro Pro Thr Pro Thr Thr Pro Ala Ala Thr Gly Ala Gly
Ala Ala 1 5 10 15Ala Ala Val Thr Pro Glu His Ala Arg Pro Arg Arg
Met Val Arg Phe 20 25 30Asn Pro Arg Ser Asp Arg Phe His Thr Leu Ser
Phe His His Val Glu 35 40 45Phe Trp Cys Ala Asp Ala Ala Ser Ala Ala
Gly Arg Phe Ala Phe Ala 50 55 60Leu Gly Ala Pro Leu Ala Ala Arg Ser
Asp Leu Ser Thr Gly Asn Ser65 70 75 80Val His Ala Ser Gln Leu Leu
Arg Ser Gly Asn Leu Ala Phe Leu Phe 85 90 95Thr Ala Pro Tyr Ala Asn
Gly Cys Asp Ala Ala Thr Ala Ser Leu Pro 100 105 110Ser Phe Ser Ala
Asp Ala Ala Arg Arg Phe Ser Ala Asp His Gly Leu 115 120 125Ala Val
Arg Ser Ile Ala Leu Arg Val Ala Asp Ala Ala Glu Ala Phe 130 135
140Arg Ala Ser Val1453094PRTAcinetobacter sp 30Met Asp Ile Leu Glu
Asn Pro Leu Glu Leu Cys Gly Phe Ala Phe Ile 1 5 10 15Glu Phe Val
Ser Lys Glu Asn Glu Leu Asp Pro Ile Phe Glu Thr Ile 20 25 30Gly Phe
Ser Lys Val Ala Lys His Lys Ser Lys Lys Ala Tyr Leu Trp 35 40 45Arg
Gln Gly Asn Ile Asn Ile Ile Leu Asn Tyr Gln Pro Glu Ser Tyr 50 55
60Ala Ser Phe Phe Phe Asn Glu His Gly Pro Ser Ala Cys Ala Met Gly65
70 75 80Phe Lys Thr Arg Asp Ala Ala Lys Ala Phe Lys Lys Ala Val 85
9031101PRTPseudomonas syringae 31 Met Ala Asp Leu Tyr Glu Ala Asp
Lys Tyr Glu Asn Pro Met Gly Leu 1 5 10 15Met Gly Phe Glu Phe Ile
Glu Phe Ala Ser Pro Thr Pro Asn Ser Leu 20 25 30Glu Pro Val Phe Gln
Met Met Gly Phe Thr Lys Val Ala Thr His Arg 35 40 45Ser Lys Asp Val
Thr Leu Tyr Arg Gln Gly Ala Ile Asn Leu Ile Leu 50 55 60Asn Asn Glu
Pro His Ser Leu Ala Ser Tyr Phe Ala Ala Glu His Gly65 70 75 80Pro
Ser Val Cys Gly Met Ala Phe Arg Val Lys Asp Ala Gln His Ala 85 90
95Tyr Asn Arg Ala Leu 1003294PRTLegionella pneumophila 32Met Gln
Asn Asn Asn Pro Cys Gly Leu Asp Gly Phe Ala Phe Leu Glu 1 5 10
15Phe Ser Gly Pro Asp Arg Asn Lys Leu His Gln Gln Phe Ser Glu Met
20 25 30Gly Phe Gln Ala Val Ala His His Lys Asn Gln Asp Ile Thr Leu
Phe 35 40 45Lys Gln Gly Glu Ile Gln Phe Ile Val Asn Ala Ala Ser His
Cys Gln 50 55 60Ala Glu Ala His Ala Ser Thr His Gly Pro Gly Ala Cys
Ala Met Gly65 70 75 80Phe Lys Val Lys Asp Ala Lys Ala Ala Phe Gln
His Ala Ile 85 903387PRTRalstonia solanacearum 33Met Ser Ala Val
Thr Thr Ala Gly Phe Ala Phe Val Glu Phe Val Cys 1 5 10 15Ala Glu
Pro Asn Glu Leu Val Ala Leu Phe Gly Lys Leu Gly Phe Lys 20 25 30Ala
Leu Gly Gln His Ala Gln Thr Gly Ala Val Leu Leu Arg Gln Asn 35 40
45Glu Ala Val Leu Ile Val Asn Pro Ala Pro Asn Pro Phe Arg Asp Val
50 55 60His Gly Ala Ser Ala Arg Ala Ile Ala Ile Asn Val Asp Asn Ala
Ala65 70 75 80Asn Ala Leu Ala Gln Ala Leu 8534130PRTBacillus
thuringiensis 34Met Val Leu Ser Met Asn His Leu Ile Tyr Leu Gln Gly
Asp Glu Asp 1 5 10 15Phe Met Lys Gln Lys Ser Met Asp Thr Leu Ala
Ala Gln Met Glu Asp 20 25 30Phe Phe Pro Val Arg Asp Val Asp His Leu
Glu Phe Tyr Val Gly Asn 35 40 45Ala Lys Gln Ser Ser Tyr Tyr Leu Ala
Arg Ala Phe Gly Phe Lys Ile 50 55 60Val Ala Tyr Ser Gly Leu Glu Thr
Gly Asn Arg Glu Lys Val Ser Tyr65 70 75 80Val Leu Val Gln Lys Asn
Met Arg Phe Val Val Ser Gly Ala Leu Ser 85 90 95Ser Asp Asn Arg Ile
Ala Glu Phe Val Lys Thr His Gly Asp Gly Val 100 105 110Lys Asp Val
Ala Leu Leu Val Asp Asp Val Asp Lys Ala Tyr Ser Glu 115 120 125Ala
Val 13035103PRTChloroflexus aurantiacus 35Met Cys Ser Ala Asp Pro
Leu Glu Leu Leu Gly Ile Asp Tyr Val Glu 1 5 10 15Phe Tyr Val Ser
Asn Ala Arg Gln Ala Ala His Phe Tyr Arg Thr Thr 20 25 30Leu Gly Leu
Arg Pro Val Ala Tyr Ala Gly Leu Glu Thr Gly Val Arg 35 40 45Asp Arg
Ala Ser Tyr Val Leu Glu Arg Arg Asn Val Arg Phe Val Leu 50 55 60Thr
Ala Pro Leu Leu Pro Asp His Pro Ile Ala Gln His Ile Ala His65 70 75
80His Gly Asp Gly Val Lys Asp Ile Ala Leu Arg Val Arg Asp Ala Val
85 90 95Thr Ala Tyr Glu Thr Ala Val 10036115PRTCatenulispora
acidphila 36Met Thr Glu Thr Ala Thr Ala Ser Ala Ala Ser Ala Thr Ala
Thr Lys 1 5 10 15Asp Pro Phe Pro Val Lys Gly Met Asp Ala Val Val
Phe Ala Val Gly 20 25 30Asn Ala Lys Gln Ala Ala His Tyr Tyr Ser Thr
Ala Phe Gly Met Arg 35 40 45Val Val Ala Tyr Ser Gly Pro Glu Thr Gly
Arg Ala Asp Arg Val Ala 50 55 60Tyr Val Leu Glu Ser Gly Ser Ala Arg
Phe Val Phe Lys Gly Ser Val65 70 75 80Arg Pro Gly Thr Glu Ile Ala
Leu His Val Ala Glu His Gly Asp Gly 85 90 95Val Thr Asp Leu Ala Ile
Ala Val Pro Asp Val Tyr Ala Ala Tyr Glu 100 105 110Tyr Ala Val
11537128PRTMicromonospora aurantiaca 37Met Thr Gln Ala Ile Asp Arg
Pro Gln Ser Thr Glu Glu Val Asp Val 1 5 10 15Asp Ala Leu Val Gly
Ala Val Asp His Asp Ile Thr Arg Asp Pro Phe 20 25 30Pro Val Lys Gly
Met Asp His Val His Phe Leu Val Gly Asn Ala Lys 35 40 45Gln Ala Ala
His Tyr Tyr Ser Thr Ala Phe Gly Met Thr Cys Val Ala 50 55 60Tyr Arg
Gly Pro Glu Gln Gly Tyr Arg Asp His Ala Gln Tyr Val Leu65 70 75
80Thr Ser Gly Ser Ala Arg Phe Val Leu Thr Gly Ala Val Arg Pro Asp
85 90 95Ala Asp Gly Ala Glu His Val Ala Lys His Ser Asp Gly Val Ser
Asp 100 105 110Ile Ala Leu Glu Val Pro Asp Val Asp Ala Ala Tyr Ala
His Ala Val 115 120 12538128PRTSalinispora tropica 38Met Thr Gln
Ala Ile Asp Arg Pro Gln Thr Ser Asp Glu Val Asp Ala 1 5 10 15Asp
Leu Leu Val Gly Ala Val Asp His Asp Ile Ser His Asp Pro Phe 20 25
30Pro Val Lys Gly Leu Asp His Val Gln Phe Leu Val Gly Asn Ala Lys
35 40 45Gln Ala Ala His Tyr Tyr Ser Thr Ala Phe Gly Met Thr Cys Val
Ala 50 55 60Tyr Arg Gly Pro Glu Gln Gly Tyr Arg Asp His Ala Gln Tyr
Val Leu65 70 75 80Thr Ser Gly Ser Ala Arg Phe Val Leu Thr Gly Ala
Val Arg Pro Asp 85 90 95Ala Ala Gly Ala Glu Gln Val Ala Arg His Ser
Asp Gly Val Cys Asp 100 105 110Ile Ala Leu Glu Val Pro Asp Val Asp
Ala Ala His Ala His Ala Ile 115 120 12539133PRTGeodermatophilus
obscurus 39Met Ser Leu Glu Gln Ala Leu Asn Asp Asp Glu Arg Leu Ala
Gln Leu 1 5 10 15Asp Leu Asp Gln Leu Lys Gln Leu Val Gly Leu Val
Glu Tyr Asp Ala 20 25 30Ser Gly Asp Pro Phe Pro Val Ser Gly Trp Asp
Ala Leu Val Trp Val 35 40 45Val Gly Asn Ala Thr Gln Ala Ala His Phe
His Gln Ser Ala Phe Gly 50 55 60Met Glu Leu Val Ala Tyr Ser Gly Pro
Glu Thr Gly Asn Arg Asp His65 70 75 80Leu Ala Tyr Val Leu Glu Ser
Gly Ala Ala Arg Phe Val Val Arg Gly 85 90 95Ala Tyr Asp Pro Ala Ser
Pro Leu Ala Asp His His Arg Lys His Gly 100 105 110Asp Gly Ile Val
Asp Ile Ala Leu Ser Val Pro Asp Val Asp Arg Cys 115 120 125Ile Ala
His Ala Ala 13040132PRTKribbella flavida 40Met Thr Ser Thr Asp Leu
Thr Pro Ala Glu Leu Asp Ala Asp Leu Asp 1 5 10 15Leu Asp Gln Leu
Lys Gln Leu Val Gly Leu Val Pro Tyr Asp Glu Ser 20 25 30Thr Asp Pro
Phe Pro Val Thr Ala Met Asp Ala Val Val Phe Val Val 35 40 45Gly Asn
Ala Thr Gln Thr Ala Lys Phe Tyr Gln Leu Ala Phe Gly Met 50 55 60Asp
Leu Val Ala Tyr Ala Gly Pro Glu Thr Gly Ser Lys Asp Ala Lys65 70 75
80Tyr Phe Val Leu Lys Ala Gly Ser Ala Arg Phe Val Ile Ser Gly Gly
85 90 95Val Arg Pro Asp Ser Pro Leu Leu Asp His His Arg Lys His Gly
Asp 100 105 110Gly Val Val Asp Leu Ala Leu Glu Val Pro Asp Val Asp
Lys Cys Val 115 120 125Lys His Ala Arg 13041117PRTStreptomyces
avermitilis 41Met Thr Gln Thr Thr His His Thr Pro Asp Thr Ala Arg
Gln Ala Asp 1 5 10 15Pro Phe Pro Val Lys Gly Met Asp Ala Val Val
Phe Ala Val Gly Asn 20 25 30Ala Lys Gln Ala Ala His Tyr Ser Thr Ala
Phe Gly Met Gln Leu Val 35 40 45Ala Tyr Ser Gly Pro Glu Asn Gly Ser
Arg Glu Thr Ala Ser Tyr Val 50 55 60Leu Thr Asn Gly Ser Ala Arg Phe
Val Leu Thr Ser Val Ile Lys Pro65 70 75 80Ala Thr Pro Trp Gly His
Phe Leu Ala Asp His Val Ala Glu His Gly 85 90 95Asp Gly Val Val Asp
Leu Ala Ile Glu Val Pro Asp Ala Arg Ala Ala 100 105 110His Ala Tyr
Ala Ile 11542142PRTOstreoccoccus tauri 42Met Thr Thr Ser Ala Ser
Gly Arg Lys Leu Val Gly His Ala Asn Phe 1 5 10 15Val Arg Cys Asn
Pro Leu Ser Asp Ala Phe Glu Cys Val Gly Phe Asp 20 25 30His Val Glu
Phe Trp Cys Gly Asp Ala Thr Asn Ala Ala Ser Arg Phe 35 40 45Gly Val
Gly Leu Gly Met Ser Leu Arg Ala Lys Ser Asp Ala Ser Thr 50 55 60Gly
Asn Gly Ile Tyr Ala Ser Tyr Ala Met Lys Ser His Asp Leu Thr65 70 75
80Phe Val Phe Thr Ala Pro Tyr Gly Asp Asp Glu Arg Ala Val Gly Cys
85 90 95Gly Gly Ser Ser Val Asn Val Pro His Pro Gly Asn Glu Arg Gly
Ala 100 105 110Met Met Arg Phe Phe Glu Arg His Gly Leu Ala Ala Arg
Ala Val Gly 115 120 125Leu Arg Val Gly Asp Ala Arg Ala Ala Tyr Glu
Glu Ala Met 130 135 14043152PRTDaucus carota 43Met Gly Lys Lys Gln
Ser Glu Ala Glu Ile Leu Ser Ser Asn Ser Ser 1 5 10 15Asn Thr Ser
Pro Ala Thr Phe Lys Leu Val Gly Phe Asn Asn Phe Val 20 25 30Arg Ala
Asn Pro Lys Ser Asp His Phe Ala Val Lys Arg Phe His His 35 40 45Ile
Glu Phe Trp Cys Gly Asp Ala Thr Asn Thr Ser Arg Arg Phe Ser 50 55
60Trp Gly Leu Gly Met Pro Leu Val Ala Lys Ser Asp Leu Ser Thr Gly65
70 75 80Asn Ser Val His Ala Ser Tyr Leu Val Arg Ser Ala Asn Leu Ser
Phe 85 90 95Val Phe Thr Ala Pro Tyr Ser Pro Ser Thr Thr Thr Ser Ser
Gly Ser 100 105 110Ala Ala Ile Pro Ser Phe Ser Ala Ser Gly Phe His
Ser Phe Ala Ala 115 120 125Lys His Gly Leu Ala Val Arg Ala Ile Ala
Leu Glu Val Ala Asp Val 130 135 140Ala Ala Ala Phe Glu Ala Ser
Val145 15044144PRTSolenostemon scutellarioides 44Met Gly Gln Glu
Ser Thr Ala Ala Ala Ala Val Val Pro Ala Glu Phe 1 5 10 15Lys Leu
Val Gly His Lys Asn Phe Val Arg Ser Asn Pro Met Ser Asp 20 25 30His
Phe Pro Val His Arg Phe His His Val Glu Phe Trp Cys Gly Asp 35 40
45Ala Thr Asn Thr Ser Arg Arg Phe Ser Trp Gly Leu Gly Met Pro Leu
50 55 60Val Ala Lys Ser Asp Leu Ser Thr Gly Asn Ser Ala His Ala Ser
Tyr65 70 75 80Leu Leu Arg Ser Gly Glu Leu Ser Phe Val Phe Thr Ala
Pro Tyr Ser 85 90 95Pro Ser Leu Ala Glu Pro Ser Ser Ala Ser Ile Pro
Thr Phe Ser Phe 100 105 110Ser Asp His Arg Ala Phe Thr Ser Ser His
Gly Leu Ala Val Arg Ala 115 120 125Val Ala Ile Gln Val Asp Ser Ala
Ser Ser Ala Tyr Ser Ala Ala Val 130 135 14045159PRTBrassica rapa
45Met Gly His Glu Asn Ala Ala Val Ser Glu Asn Gln His His Asp Asp 1
5 10 15Ala Ala Thr Thr Ser Ala Ser Pro Gly Phe Lys Leu Val Gly Phe
Ser 20 25 30Lys Phe Val Arg Lys Asn Pro Lys Ser Asp Lys Phe Lys Val
Lys Arg 35 40 45Phe His His Ile Glu Phe Trp Cys Gly Asp Ala Thr Asn
Val Ala Arg 50 55 60Arg Phe Ser Trp Gly Leu Gly Met Arg Phe Ser Ala
Lys Ser Asp Leu65 70 75 80Ser Thr Gly Asn Met Val His Ala Ser Tyr
Leu Leu Thr Ser Gly Asp 85 90 95Leu Arg Phe Leu Phe Thr Ala Pro Tyr
Ser Pro Ser Leu Ser Ala Gly 100 105 110Glu Asn Pro Pro Thr Thr Thr
Ala Ser Ile Pro Ser Phe Asp His Val 115 120 125Thr Tyr Arg Ser Phe
Phe Ser Ser His Gly Leu Gly Val Arg Ala Val 130 135 140Ala Val Glu
Val Glu Asp Ala Glu Ala Ala Phe Ser Ile Ser Val145 150
15546140PRTCoptis japonica 46Met Val Pro Ser Thr Ala Ser Asn Leu
Lys Leu Val Gly His Thr Asn 1 5 10 15Phe Val His Asn Asn Pro Lys
Ser Asp Lys Phe His Val Lys Lys Phe 20 25 30His His Ile
Glu Phe Trp Ser Thr Asp Ala Thr Asn Thr Ala Arg Arg 35 40 45Phe Ser
Trp Gly Leu Gly Met Pro Met Val Ala Lys Ser Asp Leu Ser 50 55 60Thr
Gly Asn Met Val His Ala Ser Tyr Leu Leu Arg Ser Gly Glu Leu65 70 75
80Asn Phe Leu Phe Thr Ala Pro Tyr Ser Pro Ser Ile Ala Gly Asn Thr
85 90 95Leu Thr His Thr Ala Ser Ile Pro Thr Tyr Ser His Asn Leu Ala
Arg 100 105 110Leu Phe Ala Ser Thr His Gly Leu Ala Val Arg Ala Ile
Ala Ile Glu 115 120 125Val Gln Asp Ala Glu Leu Ala Tyr Asn Ile Ser
Val 130 135 14047155PRTGlycine max 47Met Pro Ile Pro Met Cys Asn
Glu Ile Gln Ala Gln Ala Gln Ala Gln 1 5 10 15Ala Gln Ala Gln Pro
Gly Phe Lys Leu Val Gly Phe Lys Asn Phe Val 20 25 30Arg Thr Asn Pro
Lys Ser Asp Arg Phe Gln Val Asn Arg Phe His His 35 40 45Ile Glu Phe
Trp Cys Thr Asp Ala Thr Asn Ala Ser Arg Arg Phe Ser 50 55 60Trp Gly
Leu Gly Met Pro Ile Val Ala Lys Ser Asp Leu Ser Thr Gly65 70 75
80Asn Gln Ile His Ala Ser Tyr Leu Leu Arg Ser Gly Asp Leu Ser Phe
85 90 95Leu Phe Ser Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Ser Ser
Ala 100 105 110Ala Ser Ser Ala Ser Ile Pro Ser Phe Asp Ala Ala Thr
Cys Leu Ala 115 120 125Phe Ala Ala Lys His Gly Phe Gly Val Arg Ala
Ile Ala Leu Glu Val 130 135 140Ala Asp Ala Glu Ala Ala Phe Ser Ala
Ser Val145 150 15548148PRTVitis vinifera 48Met Gly Lys Gln Asn Thr
Thr Thr Asn Asn Pro Ala Pro Gly Phe Lys 1 5 10 15Leu Val Gly Phe
Ser Asn Phe Leu Arg Thr Asn Pro Met Ser Asp Arg 20 25 30Phe Gly Val
Lys Arg Phe His His Ile Glu Phe Trp Ser Thr Asp Ala 35 40 45Thr Asn
Leu Ala Arg Arg Phe Ser Trp Gly Leu Gly Met Pro Ile Val 50 55 60Ala
Lys Ser Asp Leu Ser Thr Gly Asn Val Ile His Ala Ser Tyr Leu65 70 75
80Thr Arg Ser Gly Asp Leu Asn Phe Leu Phe Thr Ala Pro Tyr Ser Pro
85 90 95Ser Ile Ala Gly Asp Leu Glu Asn Ala Ala Ala Thr Ala Ser Ile
Pro 100 105 110Ser Phe Asp His Ser Ala Cys His Ala Phe Ala Ala Ser
His Gly Leu 115 120 125Gly Val Arg Ala Ile Ala Ile Glu Val Asp Asp
Ala Glu Gly Ala Phe 130 135 140His Thr Ser Val14549146PRTMedicago
truncatula 49Met Ala Ile Glu Thr Glu Thr Gln Thr Gln Thr Gln Thr
Gly Phe Lys 1 5 10 15Leu Val Gly Phe Lys Asn Phe Val Arg Ala Asn
Pro Lys Ser Asp Arg 20 25 30Phe Asn Val Lys Arg Phe His His Val Glu
Phe Trp Cys Thr Asp Ala 35 40 45Thr Asn Thr Ala Arg Arg Phe Ser His
Gly Leu Gly Met Pro Ile Val 50 55 60Ala Lys Ser Asp Leu Ser Thr Gly
Asn Leu Thr His Ala Ser Tyr Leu65 70 75 80Leu Arg Ser Gly Asp Leu
Asn Phe Leu Phe Ser Ala Ala Tyr Ser Pro 85 90 95Ser Ile Ser Leu Ser
Ser Pro Ser Ser Thr Ala Ala Ile Pro Thr Phe 100 105 110Ser Ala Ser
Thr Cys Phe Ser Phe Ser Ala Ser His Gly Leu Ala Val 115 120 125Arg
Ala Val Ala Val Glu Val Glu Asp Ala Glu Val Ala Phe Thr Thr 130 135
140Ser Val14550111PRTHomo sapiens 50Met Thr Thr Tyr Ser Asp Lys Gly
Ala Lys Pro Glu Arg Gly Arg Phe 1 5 10 15Leu His Phe His Ser Val
Thr Phe Trp Val Gly Asn Ala Lys Gln Ala 20 25 30Ala Ser Phe Tyr Cys
Ser Lys Met Gly Phe Glu Pro Leu Ala Tyr Arg 35 40 45Gly Leu Glu Thr
Gly Ser Arg Glu Val Val Ser His Val Ile Lys Gln 50 55 60Gly Lys Ile
Val Phe Val Leu Ser Ser Ala Leu Asn Pro Trp Asn Lys65 70 75 80Glu
Met Gly Asp His Leu Val Lys His Gly Asp Gly Val Lys Asp Ile 85 90
95Ala Phe Glu Val Glu Asp Cys Asp Tyr Ile Val Gln Lys Ala Arg 100
105 11051111PRTRattus norvegicus 51Met Thr Thr Tyr Ser Asn Lys Gly
Pro Lys Pro Glu Arg Gly Arg Phe 1 5 10 15Leu His Phe His Ser Val
Thr Phe Trp Val Gly Asn Ala Lys Gln Ala 20 25 30Ala Ser Phe Tyr Cys
Asn Lys Met Gly Phe Glu Pro Leu Ala Tyr Lys 35 40 45Gly Leu Glu Thr
Gly Ser Arg Glu Val Val Ser His Val Ile Lys Gln 50 55 60Gly Lys Ile
Val Phe Val Leu Cys Ser Ala Leu Asn Pro Trp Asn Lys65 70 75 80Glu
Met Gly Asp His Leu Val Lys His Gly Asp Gly Val Lys Asp Ile 85 90
95Ala Phe Glu Val Glu Asp Cys Glu His Ile Val Gln Lys Ala Arg 100
105 11052111PRTMus musculus 52Met Thr Thr Tyr Asn Asn Lys Gly Pro
Lys Pro Glu Arg Gly Arg Phe 1 5 10 15Leu His Phe His Ser Val Thr
Phe Trp Val Gly Asn Ala Lys Gln Ala 20 25 30Ala Ser Phe Tyr Cys Asn
Lys Met Gly Phe Glu Pro Leu Ala Tyr Arg 35 40 45Gly Leu Glu Thr Gly
Ser Arg Glu Val Val Ser His Val Ile Lys Gln 50 55 60Gly Lys Ile Val
Phe Val Leu Cys Ser Ala Leu Asn Pro Trp Asn Lys65 70 75 80Glu Met
Gly Asp His Leu Val Lys His Gly Asp Gly Val Lys Asp Ile 85 90 95Ala
Phe Glu Val Glu Asp Cys Asp His Ile Val Gln Lys Ala Arg 100 105
11053111PRTBos taurus 53Met Thr Thr Tyr Ser Asp Lys Gly Glu Lys Pro
Glu Arg Gly Arg Phe 1 5 10 15Leu His Phe His Ser Val Thr Phe Trp
Val Gly Asn Ala Lys Gln Ala 20 25 30Ala Ser Tyr Tyr Cys Ser Lys Leu
Gly Phe Glu Pro Leu Ala Tyr Lys 35 40 45Gly Leu Glu Thr Gly Ser Arg
Glu Val Val Ser His Val Val Lys Gln 50 55 60Gly Gln Ile Val Phe Val
Phe Ser Ser Ala Leu Asn Pro Trp Asn Lys65 70 75 80Glu Met Gly Asp
His Leu Val Lys His Gly Asp Gly Val Lys Asp Ile 85 90 95Ala Phe Glu
Val Glu Asp Cys Asp Tyr Ile Val Gln Lys Ala Arg 100 105
11054440PRTSorghum bicolor 54Met Pro Pro Thr Pro Thr Thr Ala Ala
Ala Thr Gly Ala Ala Val Ala 1 5 10 15Ala Ala Ser Ala Glu Gln Ala
Ala Phe Arg Leu Val Gly His Arg Asn 20 25 30Phe Val Arg Val Asn Pro
Arg Ser Asp Arg Phe His Thr Leu Ala Phe 35 40 45His His Val Glu Leu
Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg 50 55 60Phe Ser Phe Gly
Leu Gly Ala Pro Leu Ala Ala Arg Ser Asp Leu Ser65 70 75 80Thr Gly
Asn Thr Ala His Ala Ser Leu Leu Leu Arg Ser Gly Ala Leu 85 90 95Ala
Phe Leu Phe Thr Ala Pro Tyr Ala His Gly Ala Asp Ala Ala Thr 100 105
110Ala Ser Leu Pro Ser Phe Ser Ala Ala Glu Ala Arg Arg Phe Ala Ala
115 120 125Asp His Gly Leu Ala Val Arg Ala Val Ala Leu Arg Val Ala
Asp Ala 130 135 140Glu Asp Ala Phe Arg Ala Ser Val Ala Ala Gly Ala
Arg Pro Ala Phe145 150 155 160Glu Pro Val Glu Leu Gly Leu Gly Phe
Arg Leu Ala Glu Val Glu Leu 165 170 175Tyr Gly Asp Val Val Leu Arg
Tyr Val Ser Tyr Pro Asp Asp Ala Asp 180 185 190Ala Ser Phe Leu Pro
Gly Phe Val Gly Val Thr Ser Pro Gly Ala Ala 195 200 205Asp Tyr Gly
Leu Arg Arg Phe Asp His Ile Val Gly Asn Val Pro Glu 210 215 220Leu
Ala Pro Ala Ala Ala Tyr Phe Ala Gly Phe Thr Gly Phe His Glu225 230
235 240Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly
Leu 245 250 255Asn Ser Met Val Leu Ala Asn Asn Ala Glu Asn Val Leu
Leu Pro Leu 260 265 270Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser
Gln Ile Gln Thr Tyr 275 280 285Leu Asp His His Gly Gly Pro Gly Val
Gln His Met Ala Leu Ala Ser 290 295 300Asp Asp Val Leu Arg Thr Leu
Arg Glu Met Gln Ala Arg Ser Ala Met305 310 315 320Gly Gly Phe Glu
Phe Met Ala Pro Pro Ala Pro Glu Tyr Tyr Asp Gly 325 330 335Val Arg
Arg Arg Ala Gly Asp Val Leu Thr Glu Ala Gln Ile Lys Glu 340 345
350Cys Gln Glu Leu Gly Val Leu Val Asp Arg Asp Asp Gln Gly Val Leu
355 360 365Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Leu
Phe Leu 370 375 380Glu Ile Ile Gln Arg Ile Gly Cys Met Glu Lys Asp
Glu Lys Gly Gln385 390 395 400Glu Tyr Gln Lys Gly Gly Cys Gly Gly
Phe Gly Lys Gly Asn Phe Ser 405 410 415Gln Leu Phe Lys Ser Ile Glu
Asp Tyr Glu Lys Ser Leu Glu Ala Lys 420 425 430Gln Ala Ala Ala Ala
Gln Gly Ser 435 4405510PRTGlycine max 55Met Pro Ile Pro Met Cys Asn
Glu Ile Gln 1 5 105610PRTGlycine max 56Met Cys Asn Glu Ile Gln Ala
Gln Ala Gln 1 5 1057488PRTGlycine max 57Met Pro Met Tyr Thr Pro Ser
Leu Ser Ala Pro Ser Ser Asn His Ile 1 5 10 15Gln Pro Ser Val Thr
Leu Pro Leu Tyr Ile Thr Thr Thr Lys Leu Asn 20 25 30Leu Lys Gln Gln
His His Thr Thr Pro Met Pro Ile Pro Met Cys Asn 35 40 45Glu Ile Gln
Ala Gln Ala Gln Ala Gln Ala Gln Pro Gly Phe Lys Leu 50 55 60Val Gly
Phe Lys Asn Phe Val Arg Thr Asn Pro Lys Ser Asp Arg Phe65 70 75
80Gln Val Asn Arg Phe His His Ile Glu Phe Trp Cys Thr Asp Ala Thr
85 90 95Asn Ala Ser Arg Arg Phe Ser Trp Gly Leu Gly Met Pro Ile Val
Ala 100 105 110Lys Ser Asp Leu Ser Thr Gly Asn Gln Ile His Ala Ser
Tyr Leu Leu 115 120 125Arg Ser Gly Asp Leu Ser Phe Leu Phe Ser Ala
Pro Tyr Ser Pro Ser 130 135 140Leu Ser Ala Gly Ser Ser Ala Ala Ser
Ser Ala Ser Ile Pro Ser Phe145 150 155 160Asp Ala Ala Thr Cys Leu
Ala Phe Ala Ala Lys His Gly Phe Gly Val 165 170 175Arg Ala Ile Ala
Leu Glu Val Ala Asp Ala Glu Ala Ala Phe Ser Ala 180 185 190Ser Val
Ala Lys Gly Ala Glu Pro Ala Ser Pro Pro Val Leu Val Asp 195 200
205Asp Arg Thr Gly Phe Ala Glu Val Arg Leu Tyr Gly Asp Val Val Leu
210 215 220Arg Tyr Val Ser Tyr Lys Asp Ala Ala Pro Gln Ala Pro His
Ala Asp225 230 235 240Pro Ser Arg Trp Phe Leu Pro Gly Phe Glu Ala
Ala Ala Ser Ser Ser 245 250 255Ser Phe Pro Glu Leu Asp Tyr Gly Ile
Arg Arg Leu Asp His Ala Val 260 265 270Gly Asn Val Pro Glu Leu Ala
Pro Ala Val Arg Tyr Leu Lys Gly Phe 275 280 285Ser Gly Phe His Glu
Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr 290 295 300Ser Glu Ser
Gly Leu Asn Ser Val Val Leu Ala Asn Asn Ser Glu Thr305 310 315
320Val Leu Leu Pro Leu Asn Glu Pro Val Tyr Gly Thr Lys Arg Lys Ser
325 330 335Gln Ile Glu Thr Tyr Leu Glu His Asn Glu Gly Ala Gly Val
Gln His 340 345 350Leu Ala Leu Val Thr His Asp Ile Phe Thr Thr Leu
Arg Glu Met Arg 355 360 365Lys Arg Ser Phe Leu Gly Gly Phe Glu Phe
Met Pro Ser Pro Pro Pro 370 375 380Thr Tyr Tyr Ala Asn Leu His Asn
Arg Ala Ala Asp Val Leu Thr Val385 390 395 400Asp Gln Ile Lys Gln
Cys Glu Glu Leu Gly Ile Leu Val Asp Arg Asp 405 410 415Asp Gln Gly
Thr Leu Leu Gln Ile Phe Thr Lys Pro Val Gly Asp Arg 420 425 430Pro
Thr Ile Phe Ile Glu Ile Ile Gln Arg Ile Gly Cys Met Val Glu 435 440
445Asp Glu Glu Gly Lys Val Tyr Gln Lys Gly Ala Cys Gly Gly Phe Gly
450 455 460Lys Gly Asn Phe Ser Glu Leu Phe Lys Ser Ile Glu Glu Tyr
Glu Lys465 470 475 480Thr Leu Glu Ala Lys Arg Thr Ala
4855886PRTGlycine max 58Met Pro Met Tyr Thr Pro Ser Leu Ser Ala Pro
Ser Ser Asn His Ile 1 5 10 15Gln Pro Ser Val Thr Leu Pro Leu Tyr
Ile Thr Thr Thr Lys Leu Asn 20 25 30Leu Lys Gln Gln His His Thr Thr
Pro Met Pro Ile Pro Met Cys Asn 35 40 45Glu Ile Gln Ala Gln Ala Gln
Ala Gln Ala Gln Pro Gly Phe Lys Leu 50 55 60Val Gly Phe Lys Asn Phe
Val Arg Thr Asn Pro Lys Ser Asp Arg Phe65 70 75 80Gln Val Asn Arg
Phe His 8559447PRTGlycine max 59Met Pro Ile Pro Met Cys Asn Glu Ile
Gln Ala Gln Ala Gln Ala Gln 1 5 10 15Ala Gln Pro Gly Phe Lys Leu
Val Gly Phe Lys Asn Phe Val Arg Thr 20 25 30Asn Pro Lys Ser Asp Arg
Phe Gln Val Asn Arg Phe His His Ile Glu 35 40 45Phe Trp Cys Thr Asp
Ala Thr Asn Ala Ser Arg Arg Phe Ser Trp Gly 50 55 60Leu Gly Met Pro
Ile Val Ala Lys Ser Asp Leu Ser Thr Gly Asn Gln65 70 75 80Ile His
Ala Ser Tyr Leu Leu Arg Ser Gly Asp Leu Ser Phe Leu Phe 85 90 95Ser
Ala Pro Tyr Ser Pro Ser Leu Ser Ala Gly Ser Ser Ala Ala Ser 100 105
110Ser Ala Ser Ile Pro Ser Phe Asp Ala Ala Thr Cys Leu Ala Phe Ala
115 120 125Ala Lys His Gly Phe Gly Val Arg Ala Ile Ala Leu Glu Val
Ala Asp 130 135 140Ala Glu Ala Ala Phe Ser Ala Ser Val Ala Lys Gly
Ala Glu Pro Ala145 150 155 160Ser Pro Pro Val Leu Val Asp Asp Arg
Thr Gly Phe Ala Glu Val Arg 165 170 175Leu Tyr Gly Asp Val Val Leu
Arg Tyr Val Ser Tyr Lys Asp Ala Ala 180 185 190Pro Gln Ala Pro His
Ala Asp Pro Ser Arg Trp Phe Leu Pro Gly Phe 195 200 205Glu Ala Ala
Ala Ser Ser Ser Ser Phe Pro Glu Leu Asp Tyr Gly Ile 210 215 220Arg
Arg Leu Asp His Ala Val Gly Asn Val Pro Glu Leu Ala Pro Ala225 230
235 240Val Arg Tyr Leu Lys Gly Phe Ser Gly Phe His Glu Phe Ala Glu
Phe 245 250 255Thr Ala Glu Asp Val Gly Thr Ser Glu Ser Gly Leu Asn
Ser Val Val 260 265 270Leu Ala Asn Asn Ser Glu Thr Val Leu Leu Pro
Leu Asn Glu Pro Val 275 280 285Tyr Gly Thr Lys Arg Lys Ser Gln Ile
Glu Thr Tyr Leu Glu His Asn 290 295 300Glu Gly Ala Gly Val Gln His
Leu Ala Leu Val Thr His Asp Ile Phe305 310 315 320Thr Thr Leu Arg
Glu Met Arg Lys Arg Ser Phe Leu Gly Gly Phe Glu 325 330 335Phe Met
Pro Ser Pro Pro Pro Thr Tyr Tyr Ala Asn Leu His Asn Arg 340 345
350Ala Ala Asp Val Leu Thr Val Asp Gln Ile Lys Gln Cys Glu Glu Leu
355 360 365Gly Ile Leu Val Asp Arg Asp Asp Gln Gly Thr Leu Leu Gln
Ile Phe 370 375 380Thr Lys Pro Val Gly Asp Arg Pro Thr Ile Phe Ile
Glu Ile Ile Gln385 390 395 400Arg Ile Gly Cys Met Val Glu Asp Glu
Glu Gly Lys Val Tyr Gln Lys
405 410 415Gly Ala Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser Glu Leu
Phe Lys 420 425 430Ser Ile Glu Glu Tyr Glu Lys Thr Leu Glu Ala Lys
Arg Thr Ala 435 440 445602064DNAGlycine max 60gtaataaaaa aagagagaag
ccgcatcaac atcatccaat atatggacgt taaaagagcg 60tcgtaatcca tttccatttc
tcatctatct tcacttcctc gtcctcatcc tcatccacct 120attctcaacc
cagacgcaat gcccatgtac actccatcac tctccgcacc ctcctccaat
180cacattcaac caagtgtcac actcccctta tatatcacaa ccaccaagct
caatctcaag 240cagcagcatc acaccacacc aatgccaata cccatgtgca
acgaaattca agcccaagcc 300caagcccaag cccaacctgg gtttaagctc
gtcggtttca aaaacttcgt ccgaaccaat 360cctaagtcgg accgctttca
agtcaaccgc ttccaccaca tcgagttctg gtgcaccgat 420gccaccaacg
cctctcgccg attctcttgg ggacttggaa tgcctattgt ggcaaaatct
480gatctctcca ccggaaacca aatccacgcc tcctacctcc tccgctccgg
cgacctctcc 540ttcctcttct ccgctcctta ctctccctct ctctccgccg
gctcctccgc tgcctcctcc 600gcctccattc ccagtttcga cgccgccacc
tgccttgcct tcgctgccaa acacggcttc 660ggcgtccgcg ccatcgcctt
ggaagtcgcc gacgcggaag ccgctttcag cgccagcgtc 720gcgaaaggag
ccgagccggc gtcgccgccg gttctcgtcg acgatcgcac cggcttcgcg
780gaggtgcgcc tctacggcga cgtggtgctc cgctacgtca gctacaagga
cgccgcgccg 840caggcgccac acgcagatcc gtcgcggtgg ttcctgccgg
gattcgaggc cgcggcgtcg 900tcgtcttcgt ttccggagct ggactacggg
atccggcggc tggaccacgc cgtcgggaac 960gttccggagc tggcgccggc
ggtgaggtac ctgaaaggct tcagcggatt ccacgagttc 1020gcggagttca
ccgcggagga cgtgggaacg agcgagagcg ggttgaactc ggtggttctg
1080gcgaacaact cggagacggt gttgctgccg ctgaacgagc cggtttacgg
aacgaagagg 1140aagagccaga ttgagacgta tttggaacac aacgaaggtg
ctggtgtgca gcaccttgcg 1200cttgttactc acgacatctt caccacactg
agagagatga gaaagcgaag tttccttggt 1260ggatttgagt tcatgccttc
tcctcctccc acctattacg ccaacctcca caaccgtgcc 1320gctgatgtgt
tgaccgttga ccagattaag cagtgtgagg agcttgggat tcttgttgac
1380agagatgatc agggcactct gcttcagatt ttcactaagc ctgttgggga
caggttcttc 1440attttctgct tctttttttt ttttttgttt ttttaatccc
tgctaaacaa ctttattata 1500actctcacat tctattagcc tagccttgat
gacttttaat ttacgttaaa ctgtgctttt 1560tattctccta ctttgttagt
ttttttttta tataaaattt taatttttca attataactt 1620tcaataatta
acaaatgatg tacagtatag tgttatgtca gagtggatgt acttgatgta
1680gcagttcatc agagtgtttc ccactacaaa ttgtactttt gtccctttcc
tgacataaag 1740tttacgacat tgaaaaaatt gatagataaa agtgcaattt
atttatcttc cgctttgaac 1800tgattgaaag tggtaaaagt tagattaaca
atttgacagt gtttgtgtgt tggagggtgg 1860tgattagtta aatgtgtttt
gtgttgaatt gacaggccaa cgatattcat agagataatt 1920cagaggatcg
ggtgcatggt ggaggatgag gaagggaagg tgtaccagaa gggtgcatgt
1980gggggttttg ggaaaggcaa tttttctgag cttttcaaat ccattgaaga
atatgagaag 2040actttggaag ctaaaagaac cgcg 2064
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References